A population modification gene drive targeting both Saglin and Lipophorin impairs Plasmodium transmission in Anopheles mosquitoes

9 figures, 2 tables and 6 additional files

Figures

Characterization and modification of the endogenous Lipophorin gene.

(A) Coomassie staining of electrophoresed hemolymph proteins. Mosquitoes were injected with the indicated double-stranded RNA to silence either Vg or Lp as described (Rono et al., 2010) and offered …

Figure 1—source data 1

Coomassie staining of an SDS-PAGE gel with electrophoresed hemolymph proteins.

Mosquitoes were injected with the indicated double-stranded RNA to silence either Vg or Lp and offered a blood meal after 2 days. Hemolymph was collected 42 hr post blood feeding.

https://cdn.elifesciences.org/articles/93142/elife-93142-fig1-data1-v3.pdf
Figure 1—source data 2

Coomassie staining of an SDS-PAGE gel with electrophoresed hemolymph proteins.

Unlabelled version of Figure 1—source data 1.

https://cdn.elifesciences.org/articles/93142/elife-93142-fig1-data2-v3.pptx
Figure 1—source data 3

Western blot with anti-ApoLpII antibody on hemolymph samples from 10 female mosquitoes.

First and 7th lanes: protein size ladder, second lane: hemolymph from WT mosquitoes. Lanes c, d: hemolymph from homozygous Lp::Sc2A10 mosquitoes. Lanes a, b: hemolymph from mosquitoes expressing a distinct Lp::ScFv fusion not further discussed in this work.

https://cdn.elifesciences.org/articles/93142/elife-93142-fig1-data3-v3.pdf
Figure 1—source data 4

Western blot with anti-ApoLpII antibody on hemolymph samples from 10 female mosquitoes.

Unlabelled version of Figure 1—source data 3.

https://cdn.elifesciences.org/articles/93142/elife-93142-fig1-data4-v3.zip
Infection status of mice bitten by Sc2A10-expressing vs. WT mosquitoes infected with Plasmodium.

Mosquito genotype and number of mice exposed to infectious mosquito bites is indicated below the bar for each condition. (A) Comparison of the percentages of infected mice after exposure to the …

Insertion of a Lipophorin suppression gene drive and its expected and unexpected outcomes.

(A) Scheme of the suppression drive construct. Upon chromosome cleavage by Cas9 (shown by scissors), homologous recombination (gray shading) via 5’ and 3’ regions of homology (HR) is expected to …

Characterization of the two types of Lp-GD integration events.

(A) The GFP-RFP line showed high homing rates when complemented with an independent vasa-Cas9 transgene. COPAS diagrams show the fluorescence of progeny neonate larvae from heterozygous GFP-RFP …

Designs of Saglin-based gene drives that also promote Lp locus modification and scheme of the chromosome conversion process.

(A) Gene drive cassette comprising Cas9 (under control of the zpg promoter) and an array of 7 gRNA-coding modules, inserted disruptively in the endogenous Saglin open reading frame on chromosome X …

Figure 5—source data 1

Western-blot using Saglin antibodies showing the absence of Saglin protein in the salivary glands of dissected SagGDvasa homozygous females (left image).

The same membrane (right image) was re-probed with serum from a human volunteer regularly bitten by mosquitoes, providing a loading control with salivary and carcass protein signals.

https://cdn.elifesciences.org/articles/93142/elife-93142-fig5-data1-v3.pdf
Figure 5—source data 2

Western-blot using Saglin antibodies showing the absence of Saglin protein in the salivary glands of dissected SagGDvasa homozygous females.

https://cdn.elifesciences.org/articles/93142/elife-93142-fig5-data2-v3.zip
Figure 5—source data 3

Western blot using serum from a human volunteer regularly bitten by mosquitoes, providing a loading control with salivary and carcass protein signals.

https://cdn.elifesciences.org/articles/93142/elife-93142-fig5-data3-v3.zip
Sag-GDzpg triggers modest homing at the Lp locus, which can be rescued by vasa-Cas9.

(A) COPAS analysis of the progeny from [SagGDzpg/+; Lp::Sc2A10/+] females crossed to WT. Weak DsRed fluorescence does not allow accurate separation of DsRed +and DsRed- larvae. Note that GFP …

Temporal dynamics of the Sag-GDvasa and Lp::Sc2A10 transgenes in 8 mosquito populations.

Transgenic mosquitoes carrying both transgenes were crossed to wild-type of the other sex (populations 1, 2, 5, 6, 7, 8) or mixed with wild-types (populations 3, 4) as indicated above the diagrams, …

Characterization of mutations in target genes.

(A, B) Characterization of Saglin failed homing mutations. A PCR product spanning the three Saglin gRNA target sites was amplified from 150 DsRed-negative larvae from generation 4 of population 1 (A)…

PCR genotyping of 31st generation individual Sag-GDvasa mosquitoes reveals deletions in the gRNA array.

(A) PCR amplicons spanning the six initial gRNAs in Sag-GDvasa were generated from 12 individual male mosquitoes (carrying a single transgene copy) from each of populations 1, 2, 5, and 7, 31 …

Figure 9—source data 1

The top part and the bottom part of each gel image was cropped to exclude empty space and re-arranged horizontally to generate Figure 9A.

For labels, see Figure 9A.

https://cdn.elifesciences.org/articles/93142/elife-93142-fig9-data1-v3.pdf
Figure 9—source data 2

The top part and the bottom part of each gel image was cropped to exclude empty space and re-arranged horizontally to generate Figure 9A.

For labels, see Figure 9A.

https://cdn.elifesciences.org/articles/93142/elife-93142-fig9-data2-v3.zip
Figure 9—source data 3

The top part and the bottom part of each gel image was cropped to exclude empty space and re-arranged horizontally to generate Figure 9A.

https://cdn.elifesciences.org/articles/93142/elife-93142-fig9-data3-v3.zip
Figure 9—source data 4

The bottom parts of these gel images was cropped to constitute the left and right panels of Figure 9C.

For labels, see Figure 9C.

https://cdn.elifesciences.org/articles/93142/elife-93142-fig9-data4-v3.pdf
Figure 9—source data 5

The bottom part this gel image was cropped to constitute the left panel of Figure 9C.

For labels, see Figure 9C.

https://cdn.elifesciences.org/articles/93142/elife-93142-fig9-data5-v3.zip
Figure 9—source data 6

The bottom part this gel image was cropped to constitute the right panel of Figure 9C.

https://cdn.elifesciences.org/articles/93142/elife-93142-fig9-data6-v3.zip

Tables

Table 1
Mass spectrometry identification of peptides from Lipophorin and Sc2A10 in hemolymph from transgenic mosquitoes and wild-type sibling controls.

The table shows the total number of peptide spectra detected in each hemolymph sample for the two Lp subunits and for Sc2A10. Hemolymph was collected from homozygous (hom) and heterozygous (het) …

SampleApoLpIApoLpIISc2A10
WT control2075 (5.8)486 (5.1)0 (0)
hom Sc2A10, sample 11627 (5.1)405 (4.8)11 (0.4)
hom Sc2A10, sample 21002 (4.4)267 (4.4)7 (0.3)
het Sc2A10, sample 11557 (10.1)351 (8.7)2 (0.1)
het Sc2A10, sample 22290 (6.0)664 (6.7)2 (0.1)
Table 2
The SagGDvasa GD shows high homing rates in the G2 generation at both the Lp and Saglin loci.

Six individual SagGDvasa G1 females mated to WT males oviposited in individual tubes and their larval progeny was scored visually for GFP and DsRed fluorescence. Homing rates are calculated as the …

Female #Total larvaeNegativesGFP + onlyDsRed + onlyGFP + DsRed +GFP inheritance and homing rate(Lp locus)DsRed inheritance rate, homing rate(Saglin locus)
18900089100 %100 %, 100%
25204048100 %92.3%, 84.6 %
35900059100 %100%, 100 %
45906053100 %89.8%, 79.6 %
55005045100 %90%, 80 %
65703054100 %94,7%, 89.4 %

Additional files

Supplementary file 1

DNA sequences of constructs used in this study.

https://cdn.elifesciences.org/articles/93142/elife-93142-supp1-v3.docx
Supplementary file 2

Tracking the evolution dynamics of Lp::Sc2A10 transgene frequency.

A parental cage was assembled containing only heterozygous transgenic mosquitoes (G0, transgene frequency = 50%). Neonate larvae of subsequent generations except G5, 6, 8, 9 (N indicates the number of larvae analysed) were analyzed using COPAS flow cytometry. Gates were drawn on COPAS diagrams around clouds of larvae corresponding to homozygous, heterozygous and negative individuals according to the intensity of GFP fluorescence, and the corresponding percentage of objects in each gate was recorded. Percentages were corrected to exclude objects not corresponding to larvae. Transgene frequency per 100 chromosomes dropped from 50% in G0 to 11.35% in G17, an average loss of 2.3% per generation.

https://cdn.elifesciences.org/articles/93142/elife-93142-supp2-v3.docx
Supplementary file 3

Fertility tests comparing the number of progeny produced by homozygous Lp::Sc2A10 female versus WT female mosquitoes.

Indicated identical numbers of virgin transgenic and WT females were mixed in cages with WT males. After blood feeding, neonate larvae produced by each cage were analyzed by flow cytometry (COPAS) and the numbers of GFP fluorescent and negative larvae were counted using WinMDI software on COPAS files. Identical fertility of the two categories of females would produce 50% of GFP positive progeny. a, b after the replicate number indicate the first and second egg batch, respectively, from the same mosquitoes. Replicate 1 was composed of smaller mosquitoes, due to higher density larval rearing. All other replicates were performed with mosquitoes of standard size. Replicate 4b was performed with older mosquitoes, with >50% of females already dead.

https://cdn.elifesciences.org/articles/93142/elife-93142-supp3-v3.docx
Supplementary file 4

Mouse parasitemia after infection by mosquito bites.

Mice are grouped by categories according to the genotype of the Plasmodium-carrying mosquitoes biting them or by the strain of parasite infecting them. Groups of 10 infected mosquito females (transgenic or their wild-type siblings grown in the same culture), were allowed to bite a mouse. Colored cells indicate the stage at which infected mice were sacrificed to prevent the appearance of malaria symptoms. n.d=not determined. ‘D’ in the third column indicates a mouse bitten by transgenic mosquitoes that showed a clearly delayed parasitemia compared to the controls. In two cases (indicated in third column), the mouse was bitten by fewer than 6 WT mosquitoes.

https://cdn.elifesciences.org/articles/93142/elife-93142-supp4-v3.xlsx
Supplementary file 5

Temporal dynamics of the Sag-GDvasa and Lp::Sc2A10 transgenes in 8 mosquito populations.

Each strip of panels on successive pages corresponds to an independent caged mosquito population. Each panel is a COPAS analysis snapshot of neonate larvae at the indicated generation, obtained as shown on the first page. Successive panels provide a global view of each transgene’s evolution on samples of 1000–4000 neonate larvae at each generation, with fluorescence intensity correlating with transgene copy number (GFP marks the Lp::Sc2A10 transgene on the Y axis, DsRed marks Sag-GDvasa on the X axis). Up to 9 partially overlapping distinct larval populations corresponding to the 9 possible genotypes (0, 1, or 2 copies of each fluorescence, sketched on page 1 and on selected panels duplicated below the original panel) can be resolved on COPAS diagrams. Dots indicated in population 1 panels correspond to debris (dead larvae, egg shells) and are difficult to distinguish from non-fluorescent larvae unless sorting them for verification under the microscope. Yellow arrow in population 2, G14 indicates the appearance of GFP negative larvae that represent a Lp homing refractory mutant (see text). Tracking Populations 1 and 2 started shortly after initial transgenesis, by crossing G2 [Sag-GDvasa / Y; Lp::Sc2A10 / +] males to WT females (considered the G0 cross for temporal tracking of genotypes). Population 3 was assembled by mixing 20 transgenics from generation 12 of population 2 with 190 WT. Population 4 was assembled by mixing 60 transgenics from generation 12 of population 2 with 189 WT. In these new G0 mixes reproductive success of transgenics proved higher than that of the WT (due to differences in the quality of the parental mosquitoes), as G1 pupae consisted of 189 GFP transgenics for 358 negatives = 34.5% instead of expected 10% (population 3) or 346 GFP transgenics for 304 negatives = 53.2% instead of expected 24.1% (population 4). In all populations, note the initial rapid convergence of genotypes towards Lp::Sc2A10 (GFP) homozygosity, the rapid initial disappearance of Sag-GDvasa (DsRed) positives lacking GFP, and the persistence of a fraction of DsRed negative larvae.

https://cdn.elifesciences.org/articles/93142/elife-93142-supp5-v3.pdf
MDAR checklist
https://cdn.elifesciences.org/articles/93142/elife-93142-mdarchecklist1-v3.pdf

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