Genomics and Applied Biology 2024, Vol.15, No.2, 107-119 http://bioscipublisher.com/index.php/gab 112 These trials highlight the importance of stepwise testing, from confined laboratory settings to more ecologically relevant environments, to ensure the safety and efficacy of gene drive technologies before field deployment (James et al., 2018; Nolan, 2020). Figure 2Agcdgene, pCO37gene-drive construct, and resulting phenotypes (Adopted from Carballar-Lejarazú et al., 2020) Image caption: (A) Agcd gene: maroon blocks, exons (E1-4); empty blocks, 3′- and 5′-untranslated regions (UTR); thick black line, introns and intergenic DNA. pCO37 plasmid: maroon blocks, homology arms from the Agcd gene; blue blocks, dominant marker gene components (3XP3 and CFP); tan blocks, drive components (nanos promoter and SpCas9 protein-encoding sequences); green blocks, guide RNA components (U6 promoter and gRNA sequence); dark gray block, φC31 attP “docking” site. Genes and features of pCO37 are not to scale and approximate sizes of components in kilobases are listed in the Materials and Methods. Recombination resulting from HDR initiated at the SpCaS9/gRNA-mediated cut site (broken-line arrow) occurring within the pink-shaded regions results in integration of the gene-drive construct. (B) CFP+ (blue arrow) and homozygous Agcd-mutant (red arrow) phenotypes in larvae and (C) Agcd-mutant (red arrow) phenotype in pupae. (D) Homozygous Agcd mutant phenotype “red eye” (red arrows) in adults. Approximate image magnifications for B, C, and D are 20, 10, and 20×, respectively (Adopted from Carballar-Lejarazú et al., 2020) 6 Advances in Delivery Methods for Gene Editing 6.1 Microinjection techniques Microinjection remains a cornerstone technique for delivering gene editing tools directly into mosquito embryos. This method, while precise, is technically challenging and labor-intensive, often requiring specialized equipment and expertise. Recent advancements have focused on improving the efficiency and applicability of microinjection. For instance, the Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) technique has been developed to deliver Cas9 ribonucleoprotein (RNP) complexes directly into the ovaries of adult female mosquitoes, bypassing the need for embryonic microinjection. This method has shown promising results in generating heritable gene edits with high efficiency across multiple mosquito species (Chaverra-Rodriguez et al., 2018; Macias et al., 2019).
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