11/12/2023 0 Comments Bioinformatics at millipore sigmaRNAi knockdown and CRISPR-Cas9 experiments have been performed in mosquito cells 26, 27, 35, 36, 37 but neither has been applied at genome-scale. Cell lines also provide a platform to propagate viruses or intracellular pathogens 30, and permit in vitro characterization of mosquito-specific drugs 31, toxins 32, viruses 33, and Wolbachia 8, 34, supporting the development of biocontrol strategies. Immune-competent mosquito cell lines are also useful in dissecting the innate immune response 27 and the unique mosquito cellular anti-viral response, which involves the somatic production of PIWI-interacting small RNAs 22, 28, 29. Studies using these cell lines have revealed dependencies, such as a need for low pH of endocytic compartments for infection 25 and specific host factors 26. These cells are most widely used to propagate and characterize mosquito-borne viruses, including dengue, yellow fever, La Crosse, Japanese encephalitis virus, West Nile, Rift Valley, o’nyong-nyong, Sindbis, and Zika viruses 24. Roughly 20 mosquito cell lines from Aedes, Culex, and Anopheles genera have been established over the last 50 years 24. Thus, to better understand the mosquito host genes involved in pathogen interactions, a method for unbiased genetic screening in mosquito cells is needed. Moreover, mosquito-borne viruses cause fewer cytopathological effects (CPE) in mosquito cells than in mammalian cells, and tend to develop persistent infections in mosquito cells but not in mammalian cells 20, 21, 22, 23. However, mosquito-borne viruses interact with a distinct set of host factors in the mammalian and insect host. CRISPR screening in mammalian cells has already provided key insights into the entry and infection mechanisms of numerous toxins, parasites, bacteria and viruses 16, 17, including mosquito-borne viruses 18, 19. This approach has transformed several areas of cell biology and revealed the function of previously unannotated genes 15. Alternative strategies under current development 6, 7 include those based on the use of endosymbiotic bacteria such as Wolbachia 8, 9, gene drives to suppress wild mosquito populations 10, 11, or introduction of disease-refractory mosquitos 12, 13, 14.Ī key advantage of the introduction of CRISPR-Cas9 technology was the ability to generate large pools of sgRNAs and simultaneously test their effect in mammalian cells. These measures are hampered by ever-increasing insecticide resistance 4, 5. Current efforts to fight malaria and other mosquito-transmitted pathogens such as dengue, Zika, Chikungunya and West Nile viruses rely on control of vector populations, mostly by means of insecticides 2, 3. Mosquito-borne diseases include a vast repertoire of viral, bacterial and parasitic diseases of medical and veterinary importance, with malaria alone causing nearly half a million human deaths each year 1. Altogether, we provide a platform for high-throughput genome-scale screening in cell lines from disease vector species. We then optimize a recombination-mediated cassette exchange system to deliver CRISPR sgRNA and perform pooled CRISPR screens in an Anopheles cell line. To develop such an approach, we design a new bioinformatic portal for sgRNA library design in several mosquito genomes, engineer mosquito cell lines to express Cas9 and accept sgRNA at scale, and identify optimal promoters for sgRNA expression in several mosquito species. An equivalent approach for genetic screening of mosquito cell lines has been lacking. Moreover, application in mammalian cells of forward genetic approaches such as CRISPR screens have identified essential genes and genes required for host-pathogen interactions, and in general, aided in functional annotation of genes. Current efforts to understand and counteract them have been aided by the use of cultured mosquito cells. Mosquito-borne diseases present a worldwide public health burden.
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