Innovative tools are essential for advancing malaria control and depend on an understanding of molecular mechanisms governing transmission of malaria parasites by Anopheles mosquitoes. CRISPR/Cas9-based gene disruption is a powerful method to uncover underlying biology of vector-pathogen interactions and can itself form the basis of mosquito control strategies. However, embryo injection methods used to genetically manipulate mosquitoes (especially Anopheles ) are difficult and inefficient, particularly for non-specialist laboratories. Here, we adapted the ReMOT Control ( Re ceptor- m ediated O vary T ransduction of C argo) technique to deliver Cas9 ribonucleoprotein complex to adult mosquito ovaries, generating targeted and heritable mutations in the malaria vector Anopheles stephensi without injecting embryos. In Anopheles , ReMOT Control gene editing was as efficient as standard embryo injections. The application of ReMOT Control to Anopheles opens the power of CRISPR/Cas9 methods to malaria laboratories that lack the equipment or expertise to perform embryo injections and establishes the flexibility of ReMOT Control for diverse mosquito species.
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ReMOT Control Delivery of CRISPR-Cas9 Ribonucleoprotein Complex to Induce Germline Mutagenesis in the Disease Vector Mosquitoes Culex pipiens pallens (Diptera: Culicidae)
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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- Award ID(s):
- 1645331
- NSF-PAR ID:
- 10228148
- Editor(s):
- Slotman, Michel
- Date Published:
- Journal Name:
- Journal of Medical Entomology
- ISSN:
- 0022-2585
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Synopsis 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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Abstract CRISPR/Cas9 gene editing is a powerful technology to study the genetics of rising model organisms, such as the jewel wasp
Nasonia 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. vitripennis females 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 thecinnabar gene 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. -
null (Ed.)The temperature-dependence of many important mosquito-borne diseases has never been quantified. These relationships are critical for understanding current distributions and predicting future shifts from climate change. We used trait-based models to characterize temperature-dependent transmission of 10 vector–pathogen pairs of mosquitoes ( Culex pipiens , Cx. quinquefascsiatus , Cx. tarsalis , and others) and viruses (West Nile, Eastern and Western Equine Encephalitis, St. Louis Encephalitis, Sindbis, and Rift Valley Fever viruses), most with substantial transmission in temperate regions. Transmission is optimized at intermediate temperatures (23–26°C) and often has wider thermal breadths (due to cooler lower thermal limits) compared to pathogens with predominately tropical distributions (in previous studies). The incidence of human West Nile virus cases across US counties responded unimodally to average summer temperature and peaked at 24°C, matching model-predicted optima (24–25°C). Climate warming will likely shift transmission of these diseases, increasing it in cooler locations while decreasing it in warmer locations.more » « less
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The Northern house mosquito (Culex pipiens) is a major vector of West Nile virus. To survive harsh conditions in winter adult females of Cx. pipiens enter a state of arrested reproductive development called diapause. Diapause is triggered by the short daylengths of late summer and early fall. The methods by which Cx. pipiens measures daylength are still unknown. However, it is suspected that clock genes, which provide information on daylength, may also regulate diapause. The proteins produced by these genes often cycle in abundance throughout the day in diapausing and nondiapausing insects. Two clock genes suspected to control diapause are cycle (cyc) and Par domain protein1 (Pdp1) as they encode circadian transcription factors that may regulate genes that are involved in diapause. Using Western blotting we measured the relative protein abundance of CYC and PDP1 throughout the day in the whole bodies and the heads of Cx. pipiens reared under either long-day, diapause-averting conditions or short-day, diapause-inducing conditions. We found that in whole bodies there was no significant oscillation of CYC or PDP1 abundance in both long day and short day-reared mosquitoes. In the heads of long day-reared mosquitoes both CYC and PDP1 cycled. In contrast, only PDP1 abundance showed diel differences in abundance in the heads of short day-reared mosquitoes. These data bring us one step closer to understanding the role that CYC and PDP1 may play in regulating diapause and other biological processes.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1093/jme/tjab016
