Journal: Science advances
Article Title: A confinable female-lethal population suppression system in the malaria vector, Anopheles gambiae .
doi: 10.1126/sciadv.ade8903
Figure Lengend Snippet: Fig. 1. We developed a transgenic crossing system to target fle for mosaic knockout that results in robust female elimination. (A) The gFLE transgene expresses two gRNAs (gRNA7 and gRNA10, teal and emerald, respectively) targeting fle near the start codon and within the first RNA recognition motif (RRM), respectively (gene map, indigo). The PolIII U6 promoter facilitates gRNA expression (navy), selection is enabled by Act-EGFP-Sv40 (purple, green, and red), and the transgene was inserted by piggyBac transgenesis [terminal repeats (TR), gray arrows]. The Cas9 transgene expresses Cas9 in the adult germline and is maternally deposited into the embryo and was previously described by Werling et al. (22). (B) Individual genotypes are identified by fluorescence with Cas9 marked by 3xP3-DsRed and gFLE marked by Act5c-GFP. (C) Crossing F0 gFLE/+ males to Cas9/+ females yields F1 offspring in Mendelian 1:1:1:1 ratios, with the gFLE/Cas9 female cohort absent. (D) The absence of all F1 gFLE/Cas9 and some gFLE/+ female offspring suggests that fle mutagenesis results in female death. This effect is heritable into the F2 and F3 generations with gFLEG/Cas9 males mated to +/+ females able to kill daughters with the appropriate genotypes (F2 and F3 data from gFLEI/Cas9 and gFLEJ/Cas9 males reported in fig. S10). (E) Crossing homozygous gFLEG/gFLEG to Cas9/Cas9 results in complete elimination of adult genetic daughters from the progeny regardless of parental sex.
Article Snippet: To identify transgene insertion sites, nanopore reads were aligned to plasmids carrying either gFLE (1154B, Addgene, as plasmid no. 187238) or Cas9 (23) constructs using minimap2 (54).
Techniques: Transgenic Assay, Knock-Out, Expressing, Selection, Fluorescence, Mutagenesis
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![Fig. 1. We developed a transgenic crossing system to target fle for mosaic knockout that results in robust female elimination. (A) The <t>gFLE</t> <t>transgene</t> expresses two gRNAs (gRNA7 and gRNA10, teal and emerald, respectively) targeting fle near the start codon and within the first RNA recognition motif (RRM), respectively (gene map, indigo). The PolIII U6 promoter facilitates gRNA expression (navy), selection is enabled by Act-EGFP-Sv40 (purple, green, and red), and the transgene was inserted by piggyBac transgenesis [terminal repeats (TR), gray arrows]. The Cas9 transgene expresses Cas9 in the adult germline and is maternally deposited into the embryo and was previously described by Werling et al. (22). (B) Individual genotypes are identified by fluorescence with Cas9 marked by 3xP3-DsRed and gFLE marked by Act5c-GFP. (C) Crossing F0 gFLE/+ males to Cas9/+ females yields F1 offspring in Mendelian 1:1:1:1 ratios, with the gFLE/Cas9 female cohort absent. (D) The absence of all F1 gFLE/Cas9 and some gFLE/+ female offspring suggests that fle mutagenesis results in female death. This effect is heritable into the F2 and F3 generations with gFLEG/Cas9 males mated to +/+ females able to kill daughters with the appropriate genotypes (F2 and F3 data from gFLEI/Cas9 and gFLEJ/Cas9 males reported in fig. S10). (E) Crossing homozygous gFLEG/gFLEG to Cas9/Cas9 results in complete elimination of adult genetic daughters from the progeny regardless of parental sex.](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_6109/pm37406109/pm37406109__page3_image1.jpg)