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1. ReMOT Control Delivery of CRISPR-Cas9 Ribonucleoprotein Complex to Induce Germline Mutagenesis in the Disease Vector Mosquitoes Culex pipiens pallens (Diptera: Culicidae)

   https://doi.org/10.1093/jme/tjab016  

   Li, Xixi ; Xu, Yang ; Zhang, Hongbo ; Yin, Haitao ; Zhou, Dan ; Sun, Yan ; Ma, Lei ; Shen, Bo ; Zhu, Changliang ( February 2021 , Journal of Medical Entomology)

   Slotman, Michel (Ed.)

   Abstract The wide distribution of Culex (Cx.) pipiens complex mosquitoes makes it difficult to prevent the transmission of mosquito-borne diseases in humans. Gene editing using CRISPR/Cas9 is an effective technique with the potential to solve the growing problem of mosquito-borne diseases. This study uses the ReMOT Control technique in Culex pipiens pallens (L.) to produce genetically modified mosquitoes. A microinjection system was established by injecting 60 adult female mosquitoes—14 µl injection mixture was required, and no precipitation occurred with ≤1 µl of endosomal release reagents (chloroquine or saponin). The efficiency of delivery of the P2C-enhanced green fluorescent protein-Cas9 (P2C-EGFP-Cas9) ribonucleoprotein complex into the ovary was 100% when injected at 24 h post-bloodmeal (the peak of vitellogenesis). Using this method for KMO knockout, we found that gene editing in the ovary could also occur when P2C-Cas9 RNP complex was injected into the hemolymph of adult Cx. pipiens pallens by ReMOT Control. In the chloroquine group, of the 2,251 G0 progeny screened, 9 individuals showed with white and mosaic eye phenotypes. In the saponin group, of the 2,462 G0 progeny screened, 8 mutant individuals were observed. Sequencing results showed 13 bp deletions, further confirming the fact that gene editing occurred. In conclusion, the successful application of ReMOT Control in Cx. pipiens pallens not only provides the basic parameters (injection parameters and injection time) for this method but also facilitates the study of mosquito biology and control. 

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2. The Development and Expansion of in vivo Germline Editing Technologies in Arthropods: Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) and Beyond

   https://doi.org/10.1093/icb/icad123  

   Terradas, Gerard ; Macias, Vanessa M. ; Peterson, Hillary ; McKeand, Sage ; Krawczyk, Grzegorz ; Rasgon, Jason L. ( September 2023 , Integrative And Comparative Biology)

   In the past 20 years, sequencing technologies have led to easy access to genomic data from nonmodel organisms in all biological realms. Insect genetic manipulation, however, continues to be a challenge due to various factors, including technical and cost-related issues. Traditional techniques such as microinjection of gene-editing vectors into early stage embryos have been used for arthropod transgenesis and the discovery of Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein (CRISPR–Cas) technologies allowed for targeted mutagenesis and the creation of knockouts or knock-ins in arthropods. Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) acts as an alternative to embryonic microinjections, which require expensive equipment and extensive hands-on training. ReMOT Control’s main advantage is its ease of use coupled with the ability to hypothetically target any vitellogenic species, as injections are administered to the egg-laying adult rather than embryos. After its initial application in the mosquito Aedes aegypti, ReMOT Control has successfully produced mutants not only for mosquitoes but for multiple arthropod species from diverse orders, such as ticks, mites, wasps, beetles, and true bugs, and is being extended to crustaceans, demonstrating the versatility of the technique. In this review, we discuss the current state of ReMOT Control from its proof-of-concept to the advances and challenges in the application across species after 5 years since its development, including novel extensions of the technique such as direct parental (DIPA)-CRISPR.

    

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3. Transcriptional response of individual Hawaiian Culex quinquefasciatus mosquitoes to the avian malaria parasite Plasmodium relictum

   https://doi.org/10.1186/s12936-022-04271-x  

   Ferreira, Francisco C. ; Videvall, Elin ; Seidl, Christa M. ; Wagner, Nicole E. ; Kilpatrick, A. Marm ; Fleischer, Robert C. ; Fonseca, Dina M. ( August 2022 , Malaria Journal)

   Plasmodiumparasites that cause bird malaria occur in all continents except Antarctica and are primarily transmitted by mosquitoes in the genusCulex.Culex quinquefasciatus, the mosquito vector of avian malaria in Hawaiʻi, became established in the islands in the 1820s. While the deadly effects of malaria on endemic bird species have been documented for many decades, vector-parasite interactions in avian malaria systems are relatively understudied.

   To evaluate the gene expression response of mosquitoes exposed to aPlasmodiuminfection intensity known to occur naturally in Hawaiʻi, offspring of wild-collected HawaiianCx. quinquefasciatuswere fed on a domestic canary infected with a fresh isolate ofPlasmodium relictumGRW4 from a wild-caught Hawaiian honeycreeper. Control mosquitoes were fed on an uninfected canary. Transcriptomes of five infected and three uninfected individual mosquitoes were sequenced at each of three stages of the parasite life cycle: 24 h post feeding (hpf) during ookinete invasion; 5 days post feeding (dpf) when oocysts are developing; 10 dpf when sporozoites are released and invade the salivary glands.

   Differential gene expression analyses showed that during ookinete invasion (24 hpf), genes related to oxidoreductase activity and galactose catabolism had lower expression levels in infected mosquitoes compared to controls. Oocyst development (5 dpf) was associated with reduced expression of a gene with a predicted innate immune function. At 10 dpf, infected mosquitoes had reduced expression levels of a serine protease inhibitor, and further studies should assess its role as aPlasmodiumagonist inC. quinquefasciatus. Overall, the differential gene expression response of HawaiianCulexexposed to aPlasmodiuminfection intensity known to occur naturally in Hawaiʻi was low, but more pronounced during ookinete invasion.

   This is the first analysis of the transcriptional responses of vectors to malaria parasites in non-mammalian systems. Interestingly, few similarities were found between the response ofCulexinfected with a birdPlasmodiumand those reported inAnophelesinfected with humanPlasmodium. The relatively small transcriptional changes observed in mosquito genes related to immune response and nutrient metabolism support conclusions of low fitness costs often documented in experimental challenges ofCulexwith avianPlasmodium.

    

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4. Novel genome sequences and evolutionary dynamics of the North American anopheline species Anopheles freeborni , Anopheles crucians , Anopheles quadrimaculatus , and Anopheles albimanus

   https://doi.org/10.1093/g3journal/jkac284  

   Henderson, Cory ; Kemirembe, Karen ; McKeand, Sage ; Bergey, Christina ; Rasgon, Jason L. ; Kern, ed., A. ( November 2022 , G3)

   Anopheles mosquitoes are the principal vectors for malaria and lymphatic filariasis, and evidence for arboviral transmission under laboratory and natural contexts has been demonstrated. Vector management approaches require an understanding of the ecological, epidemiological, and biological contexts of the species in question, and increased interest in gene drive systems for vector control applications has resulted in an increased need for genome assemblies from understudied mosquito vector species. In this study, we present novel genome assemblies for Anopheles crucians, Anopheles freeborni, Anopheles albimanus, and Anopheles quadrimaculatus and examine the evolutionary relationship between these species. We identified 790 shared single-copy orthologs between the newly sequenced genomes and created a phylogeny using 673 of the orthologs, identifying 289 orthologs with evidence for positive selection on at least 1 branch of the phylogeny. Gene ontology terms such as calcium ion signaling, histone binding, and protein acetylation identified as being biased in the set of selected genes. These novel genome sequences will be useful in developing our understanding of the diverse biological traits that drive vectorial capacity in anophelines.

    

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5. Germline mutagenesis of Nasonia vitripennis through ovarian delivery of CRISPR‐Cas9 ribonucleoprotein

   https://doi.org/10.1111/imb.12663  

   Chaverra‐Rodriguez, D. ; Dalla Benetta, E. ; Heu, C. C. ; Rasgon, J. L. ; Ferree, P. M. ; Akbari, O. S. ( August 2020 , Insect Molecular Biology)

   CRISPR/Cas9 gene editing is a powerful technology to study the genetics of rising model organisms, such as the jewel waspNasonia vitripennis. However, current methods involving embryonic microinjection of CRISPR reagents are challenging. Delivery of Cas9 ribonucleoprotein into female ovaries is an alternative that has only been explored in a small handful of insects, such as mosquitoes, whiteflies and beetles. Here, we developed a simple protocol for germline gene editing by injecting Cas9 ribonucleoprotein in adultN. vitripennisfemales using either ReMOT control (Receptor‐Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules) as ovary delivery methods. For ReMOT Control we used theDrosophila melanogaster‐derived peptide ‘P2C’ fused to EGFP to visualize the ovary delivery, and fused to Cas9 protein for gene editing of thecinnabargene using saponin as an endosomal escape reagent. For BAPC we optimized the concentrations of protein, sgRNA and the transfection reagent. We demonstrate delivery of protein cargo such as EGFP and Cas9 into developing oocytes via P2C peptide and BAPC. Additionally, somatic and germline gene editing were demonstrated. This approach will greatly facilitate CRISPR‐applied genetic manipulation in this and other rising model organisms.

    

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