Journal of Mosquito Research 2024, Vol.14, No.3, 124-134 http://emtoscipublisher.com/index.php/jmr 126 the use of HEGs, which can spread rapidly through a population and disrupt essential genes, leading to population decline (Windbichler et al., 2011).These suppression strategies have shown potential in both laboratory and field trials, although their long-term ecological impacts require further study. 3.3 Population replacement Population replacement strategies aim to replace wild mosquito populations with genetically modified ones that are less capable of transmitting diseases. This can be achieved by introducing genes that confer resistance to pathogens such as malaria or dengue virus. Gene drive systems, particularly those based on CRISPR/Cas9 technology, have been developed to enhance the inheritance of these resistance genes, ensuring that they spread rapidly through the population. For instance, gene-drive rescue systems have been designed to maintain the functionality of essential genes while spreading the desired modifications, thereby ensuring the survival and propagation of the modified mosquitoes (Figure 1) (Adolfi et al., 2020). These strategies hold promise for long-term disease control, but their implementation requires careful consideration of potential ecological and evolutionary consequences (Nazareth et al., 2020). In summary, genetic control techniques offer innovative solutions for mosquito population management through genetic modification, population suppression, and population replacement. Each approach has its unique mechanisms and potential impacts, necessitating thorough evaluation and monitoring to ensure their efficacy and safety in real-world applications (Alphey and Alphey, 2014). Figure 1 Efficient population modification gene-drive rescue system in the malaria mosquito Anopheles stephensi (Adopted from Adolfi et al., 2020) Image caption: a Swap strategy for Cas9/gRNA-mediated cassette exchange. Two plasmid-encoded gRNAs (top) guide cleavage in the genome of the white-eyed nRec mosquito line (khnRec–)8 (middle), leading to the excision of a fragment including the DsRed eye (3xP3) marker and the two antimalarial effectors m2A10 and m1C311. The HDR template plasmid (bottom) carries homology arms flanking either cut site, promoting the insertion of a GFP-marked donor template that carries a recoded portion of the kh gene followed by the 3′-end sequence of the An. gambiae kh gene including the 3′UTR (A.gam.3′) to minimize homology. b The insertion of this unit restores kh gene function while creating a sequence (khRec+) that is uncleavable by the endogenous drive components. c The Reckh gene-drive includes an An. stephensi codon-optimized Cas9 driven by the germline-specific vasa promoter from An. stephensi and a gRNA (gRNA-kh2) directed to the fifth exon of the unmodified kh+ gene (top) regulated by the ubiquitous promoter of the An. stephensi U6A gene8. The cut in the kh gene of the Reckh mosquito germline can be repaired by drive integration via HDR (homology-directed repair) or by the less desirable EJ (end-joining) pathway (bottom). HDR results in the integration of the drive cassette that maintains kh gene function at the integration site (khRec+), while EJ usually causes the formation of loss-of-function alleles (kh−). When function is lost in both copies of the gene, individuals with white eyes are produced. kh, kynurenine hydroxylase gene; attP, φC31 recombination site; U6A, RNA polymerase-III promoter; gRNA, guide RNAs; Cas9, Cas9 open reading frame; vasa, vasa promoter; 3xP3, eye-marker promoter; GFP, green fluorescent protein; dominant marker gene. The horizontal dimension of the mosquito heads at the eyes in the images is ~1 mm (Adapted from Adolfi et al., 2020)
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