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. 2015 Dec 8;112(49):E6736-43.
doi: 10.1073/pnas.1521077112. Epub 2015 Nov 23.

Highly Efficient Cas9-mediated Gene Drive for Population Modification of the Malaria Vector Mosquito Anopheles Stephensi

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Free PMC article

Highly Efficient Cas9-mediated Gene Drive for Population Modification of the Malaria Vector Mosquito Anopheles Stephensi

Valentino M Gantz et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Genetic engineering technologies can be used both to create transgenic mosquitoes carrying antipathogen effector genes targeting human malaria parasites and to generate gene-drive systems capable of introgressing the genes throughout wild vector populations. We developed a highly effective autonomous Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9)-mediated gene-drive system in the Asian malaria vector Anopheles stephensi, adapted from the mutagenic chain reaction (MCR). This specific system results in progeny of males and females derived from transgenic males exhibiting a high frequency of germ-line gene conversion consistent with homology-directed repair (HDR). This system copies an ∼ 17-kb construct from its site of insertion to its homologous chromosome in a faithful, site-specific manner. Dual anti-Plasmodium falciparum effector genes, a marker gene, and the autonomous gene-drive components are introgressed into ∼ 99.5% of the progeny following outcrosses of transgenic lines to wild-type mosquitoes. The effector genes remain transcriptionally inducible upon blood feeding. In contrast to the efficient conversion in individuals expressing Cas9 only in the germ line, males and females derived from transgenic females, which are expected to have drive component molecules in the egg, produce progeny with a high frequency of mutations in the targeted genome sequence, resulting in near-Mendelian inheritance ratios of the transgene. Such mutant alleles result presumably from nonhomologous end-joining (NHEJ) events before the segregation of somatic and germ-line lineages early in development. These data support the design of this system to be active strictly within the germ line. Strains based on this technology could sustain control and elimination as part of the malaria eradication agenda.

Keywords: CRISPR; MCR; Plasmodium falciparum; eradication; transgenesis.

Conflict of interest statement

Conflict of interest statement: E.B. and V.G. are authors of a patent applied for by the University of California, San Diego that relates to the mutagenic chain reaction.

Figures

Fig. 1.
Fig. 1.
Site-specific integration into the An. stephensi kynurenine hydroxylasewhite locus of the gene-drive construct AsMCRkh2, carrying antimalarial effector genes. (A) Schematic representations of the kynurenine hydroxylasewhite locus and AsMCRkh2 construct. Genes and other features of the AsMCRkh2 construct are not to scale. The dark red boxes represent the eight exons of the endogenous khw gene locus (Top) with the direction of transcription indicated by the wedge in exon 8. The black lines represent genomic and intron DNA. The green arrowhead represents the target site of the gRNA, kh2. Labels and arrows indicate names, approximate positions, and directions of oligonucleotide primers used in the study. khw gene sequences corresponding to previously characterized mutations are indicated as an orange rectangle (28) and square (29). The plasmid, AsMCRkh2 (Bottom), carries promoter and coding sequences comprising vasa-Cas9 and the U6A-kh2 gRNA genes (U6A gRNA) linked to the dual scFv antibody cassette (m2A10-m1C3) conferring resistance to P. falciparum (11) and the dominant eye marker gene (DsRed) inserted between regions of homology (dark red boxes) from the An. stephensi khw locus that directly abut the U6A-kh2 gRNA cut site. The black lines represent khw intron sequences, and the gray lines indicate plasmid DNA sequences. Following gRNA-directed cleavage by the Cas9–kh2 gRNA nuclease complex at the kh2 target site (green arrowhead), homology-directed repair (HDR) leads to precise insertion of the AsMCRkh2 cargo (m2A10-m1C3, DsRed, vasa-Cas9, U6A gRNA) into the genomic khw locus via HDR events somewhere within the regions of homology (pink-shaded quadrilaterals). Plasmid sequences are not integrated. (B) Gene amplification analysis confirms integration of the AsMCRkh2 cargo in genomic DNA prepared from the two G1 male transformants (10.1 and 10.2) that were positive for the DsRed eye-marker phenotype. Both males carry left and right junction fragments of the AsMCRkh2 cargo with the supplied khw regions of homology (KM1F1/Vg5′R1 and U6F1/KM2R1 primer combinations, respectively). An amplicon corresponding to the wild-type khw locus (505/557 primer pairs) confirms that these mosquitoes were heterozygous in some of their cells. Wild-type (wt) control DNA supports amplification only of the wild-type khw locus (505/557 primers). (C) Gene amplification analysis confirms site-specific integration of the AsMCRkh2 construct at the khw locus using primers located outside of the genomic sequence included in the AsMCRkh2 cassette (the left integration junction fragment amplified with primers G1F2/Vg5′R2, and the right junction fragment amplified with primers U6F1/G2R2). Wild-type control DNA did not support amplification of these hybrid fragments. Numbers refer to the length in nucleotides of the amplified fragments. Amplicon primary structure was verified by DNA sequencing (SI Appendix, Fig. S1).
Fig. 2.
Fig. 2.
Larval and adult phenotypes of AsMCRkh2 transgenic An. stephensi. Bright-field and fluorescent images of larval (A and B) and adult (C and D) eye-color phenotypes. All images are lateral views of the head. Phenotypic descriptions are listed above. White arrows in the larval images indicate the white-eye phenotype, and the yellow arrow indicates the wild-type eye color. Note that all data presented in Tables 1 and 2 for the DsRed+ phenotype are from scoring larvae, not adults. The white arrow in the adult images indicates a patch of wild-type cells in a white-eye background of the left mosaic. The right mosaic exemplifies the colored-eye phenotype.
Fig. 3.
Fig. 3.
Phenotypic inheritance patterns of the AsMCRkh2 gene-drive cargo. (Top) DsRed-positive G2 transgenic adult males and females with wild-type eye color (DsRed+/khw+, half-red and half-black circles) were outcrossed to wild-type mosquitoes (all-black circles) of the opposite sex. (Middle, Upper) G3 progeny resulting from the male outcrosses were predominantly DsRed+/khw+ (half-red and half-black circles; Table 1). G3 progeny resulting from the female outcrosses were predominantly positive for DsRed and had white (DsRed+/khw−, half-red and half-white circles) or mosaic eyes (DsRed+/mosaic, half-red and half-checkered white and black circles). (Middle, Lower) DsRed+/khw+ (half-red and half-black circles) and DsRed+/khw− (half-red and half-white circles) G3 adult males and females were outcrossed to wild-type mosquitoes (all-black circles) of the opposite sex. Specific crosses and tables for the data are referenced. (Bottom) G4 progeny resulting from outcrosses of DsRed+/khw+ G3 adult males (half-red and half-black circles; crosses 6 and 8) were predominantly DsRed+/khw+ (half-red and half-black circles), whereas those from G3 DsRed+/khw+ adult females (half-red and half-black circles; crosses 5 and 7) were predominantly positive for DsRed and had white (DsRed+/khw−, half-red and half-white circles) or mosaic eyes (DsRed+/mosaic, half-red and half-checkered white and black circles). In contrast, G4 progeny resulting from outcrosses of DsRed+/khw− G3 adult males (crosses 2 and 4) were either DsRed+/khw+ (half-red and half-black circles) or DsRed/khw+ (wild-type eye, all-black circles). G4 progeny derived from female DsRed+/khw− outcrosses (crosses 1 and 3) were also a mix of DsRed+ and DsRed. Nearly all DsRed+ progeny had white (DsRed+/khw−, half-red and half-white circles) or mosaic (DsRed+/mosaic, half-red and half checkered circles) eyes (Table 1). Among the DsRed progeny, approximately half had wild-type eyes (all-black circles) and half had white eyes (half-black and half-white circles). Male-derived (G2 cross) G4 progeny (Left) show a bias of HDR over NHEJ, whereas female-derived (G2 cross) G4 progeny lines display nearly equal HDR and NHEJ.
Fig. 4.
Fig. 4.
Expression of m1C3 and m2A10 transcripts in AsMCRkh2 transgenic females. RT-PCR was used to detect m1C3 (AgCPA-m1C3) and m2A10 (AsVg1-m2A10) transcripts in RNA isolated from homogenates of dissected midguts and the remaining carcasses of mixed heterozygous and homozygous DsRed+ G3 females at 0 (non–blood-fed), 4, 12, 24, and 48 h post blood meal. Male transgenic mosquitoes were used as negative controls. The An. stephensi S26 ribosomal protein transcript was amplified from all samples as a loading control.
Fig. 5.
Fig. 5.
Model of AsMCRkh2 transgene activity in adult males and females. (Top) Schematic representations of the third chromosomes of An. stephensi. Transgenic males (Left) and females (Right) are depicted as being homozygous in the germline for AsMCRkh2 (red bars) and are outcrossed to wild-type mosquitoes of the opposite sex. Zygotes resulting from outcrosses of transgenic males do not have the Cas9 nuclease in the eggs (clear oval), which are derived from wild-type females, and somatic cells remain heterozygous for the AsMCRkh2 transgene. A schematic representation of the sperm attached to the egg and the donated paternal chromosome is represented encircled by the dotted line. vasa-mediated expression of Cas9 is restricted to the germ line (colored half-oval) in developing embryos derived from transgenic AsMCRkh2 males, resulting in significant HDR (red arrowhead) that converts the majority of the chromosomes by insertion of the AsMCRkh2 cargo. Adults are phenotypically positive for the dominant reporter gene, DsRed, and wild-type in eye color. In contrast, zygotes resulting from outcrosses of transgenic females have Cas9 nuclease in the eggs (aqua-colored oval) as a result of vasa-directed expression in the maternal germ line, and this catalyzes nonhomologous end joining (asterisk) to mutate the paternally derived wild-type chromosome (encircled by the dotted line). Some HDR may occur at this stage, but may be hampered by an initial physical separation of the maternal and paternal chromosomes. Embryos derived from transgenic AsMCRkh2 females also have vasa-mediated Cas9 expression restricted to the germ line (colored half-oval), but in addition have the nuclease perduring from the maternal gamete (light-colored half-oval), which can result in adults that are phenotypically positive for the dominant reporter gene, DsRed, and exhibit the white or mosaic eye color. Furthermore, the paternally derived chromosomes mutagenized in the zygotes are resistant to subsequent HDR and insertion of the cargo. Some of the male-derived chromosomes may not be mutagenized, and these can be substrates for HDR. Both options are shown as the asterisk overlying the red bar in the germ line.

Comment in

  • Driving out malaria.
    Nawy T. Nawy T. Nat Methods. 2016 Feb;13(2):111. doi: 10.1038/nmeth.3755. Nat Methods. 2016. PMID: 27243093 No abstract available.

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