A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae

Journal name:
Nature Biotechnology
Volume:
34,
Pages:
78–83
Year published:
DOI:
doi:10.1038/nbt.3439
Received
Accepted
Published online

Abstract

Gene drive systems that enable super-Mendelian inheritance of a transgene have the potential to modify insect populations over a timeframe of a few years. We describe CRISPR-Cas9 endonuclease constructs that function as gene drive systems in Anopheles gambiae, the main vector for malaria. We identified three genes (AGAP005958, AGAP011377 and AGAP007280) that confer a recessive female-sterility phenotype upon disruption, and inserted into each locus CRISPR-Cas9 gene drive constructs designed to target and edit each gene. For each targeted locus we observed a strong gene drive at the molecular level, with transmission rates to progeny of 91.4 to 99.6%. Population modeling and cage experiments indicate that a CRISPR-Cas9 construct targeting one of these loci, AGAP007280, meets the minimum requirement for a gene drive targeting female reproduction in an insect population. These findings could expedite the development of gene drives to suppress mosquito populations to levels that do not support malaria transmission.

At a glance

Figures

  1. Gene disruption by homology-directed repair (HDR) at three separate loci causes recessive female sterility.
    Figure 1: Gene disruption by homology-directed repair (HDR) at three separate loci causes recessive female sterility.

    (a) A plasmid-based source of either a TALEN or Cas9 coupled with a gRNA induces a DSB at the target locus. A plasmid (hdrGFP) containing regions of homology immediately upstream and downstream of the cut site acts as a template for homology-directed repair. Internal to the homology regions a 3xP3::GFP cassette identifies hdrGFP integration events and two attP sites facilitate secondary modification of the locus through RMCE. (b) PCR was used to confirm the targeted loci in WT individuals as well as those homozygous and heterozygous for the hdrGFP allele. The primer pair used is indicated in a (blue arrows). (c) Counts of larval progeny from individual females homozygous or heterozygous for hdrGFP alleles mated to WT males. Heterozygous docking lines for all three loci showed at least full fertility compared to WT females. A minimum of 20 individuals were tested for each line. Vertical bars represent the mean and error bars the s.e.m.

  2. CRISPRh alleles inserted at female-fertility loci show highly efficient gene drive activity and can spread in a caged population.
    Figure 2: CRISPRh alleles inserted at female-fertility loci show highly efficient gene drive activity and can spread in a caged population.

    (a) RMCE was used to replace the GFP transcription unit in hdrGFP docking lines with a CRISPR homing construct (CRISPRh consisting of a 3xP3::RFP marker, Cas9 under the transcriptional control of the vasa2 promoter and a gRNA under the control of the ubiquitous U6 PolIII. The gRNA cleaves at the nondisrupted WT allele. Repair of the cleaved chromosome through HDR leads to copying of the CRISPRh allele and homing. (b) Confinement of homing to the germline should lead to super-Mendelian inheritance of a homing construct (indicated in red) that, when targeting a haplosufficient, somatic female-fertility gene, will reduce the number of fertile females. (c) High levels of homing at all three female-fertility loci were observed. Male or female CRISPRh/+ heterozygotes were mated to WT. Progeny from individual heterozygous females were scored for the presence of the RFP linked to the CRISPRh construct and the average transmission rate indicated by vertical bar (± s.e.m.). A minimum of 34 females were analyzed for each cross. The average homing rate is also shown. (d,e) Counts of eggs and hatching larvae for the individual crosses revealed a strong fertility effect in heterozygous CRISPRh/+ females (d) that was not seen in equivalent heterozygous males (e). (f) Dynamics calculated using recurrence equations in Deredec et al.10, using the observed homing rates in males and females and effects on female fertility. We assume no fitness effects in males and that the initial release consists of heterozygous males equal to 10% of the prerelease adult male population (i.e., 5% of the overall population). The model assumes discrete generations (one per month) and random mating; results are plotted starting from the first generation after release and do not account for evolution of either the CRISPR allele or the target sequence. (g) Increase in frequency of CRISPRh allele in cage population experiments. An equal number of CRISPRh/+ and WT individuals were used to start a population, and the frequency of individuals containing a CRISPRh allele was recorded in each subsequent generation. Black line shows deterministic prediction based on observed parameter values (homing rates 98.4%, heterozygous female fitness of 9.3%, homozygous females completely sterile), assuming no fitness effects in males. Gray lines show results from 20 stochastic simulations assuming 300 males and 300 females are used to start the next generation, females mate randomly with a single male and 15% of females fail to mate, using random numbers drawn from the appropriate multinomial distributions. Red lines show results from two replicate cages.

  3. Molecular confirmation ofCRISPR-induced HDR at 3 candidate female fertility loci
    Supplementary Fig. 1: Molecular confirmation ofCRISPR–induced HDR at 3 candidate female fertility loci

    A PCR was performed using a primer pair (blue arrows) specific for either the 5′ or 3′ side of the putative integration of the hdrGFP docking construct. In each case a primer binding internal to the hdrGFP construct was combined with a primer binding external to the region of homology included in the hdrGFP plasmid such that an amplicon of the expected size should only be produced in the event of the precise homol-ogy-dependent repair event illustrated. 3 individuals of each line were tested.

  4. Docking lines containing HDR gene disruptions show sterility at different stages of egg maturation.
    Supplementary Fig. 2: Docking lines containing HDR gene disruptions show sterility at different stages of egg maturation.

    Females homozygous and heterozygous for the hdrGFP allele were mated in groups to wild type males and al-lowed to lay individually and the number of resulting eggs and larvae counted. A minimum of 20 individuals were tested for each line.

  5. Molecular confirmation of CRISPRh alleles at candidate female fertility loci
    Supplementary Fig. 3: Molecular confirmation of CRISPRh alleles at candidate female fertility loci

    Following RMCE with a CRISPRh construct a PCR was performed to confirm the molecular nature of the event using a primer pair (blue arrows) designed to amplify from the docking site in the respective gene (blue bar) to an internal region of the CRISPRh construct. Primer pairs were designed for both the 5′ and 3′ ends of the integration. Nature Biotechnology

  6. NHEJ and non-canonical CRISPR induced homing events at target loci
    Supplementary Fig. 4: NHEJ and non–canonical CRISPR induced homing events at target loci

    32 progeny from CRISPRh/+ parents that did not show the RFP+ phenotype associated with the CRISPRh con-struct were analysed. The sequence included in the protospacer of the guide RNA is highlighted, with the PAM region in blue. Across the 3 loci we recovered 15 instances of mutations at the target loci and 17 instances showing wild type sequences. Among the mutations we recovered, 6 unique (originating from separate parents) NHEJ events resulting in indels and one unique incomplete homing event at AGAP007280 that incorporated a small region surrounding the guide RNA in the homing construct (lower panel).

  7. Somatic activity of vasa::Cas9
    Supplementary Fig. 5: Somatic activity of vasa::Cas9

    Progeny from a homozygous vasa::Cas9;3XP3::YFP male crossed to a heterozygous U6::gRNA(YFP) female show variegated YFP expression in somatic tissues, including eye, dorsal ganglion and anal palps (left) as well as extensive mottling throughout the body (right). The two phenotypes segregated approximately 50:50, consis-tent with somatic expression of vasa::Cas9 in all individuals and Mendelian segregation of the gRNA allele to half the progeny. Larvae were visualised under a GFP filter.

  8. Schematic of the three target genes used in this study and the nuclease binding sequences.
    Supplementary Fig. 6: Schematic of the three target genes used in this study and the nuclease binding sequences.

    The CRISPR target sequences are highlighted in grey; each includes a protospacer adjacent motif (PAM) se-quence at the target locus (5′-NGG), highlighted in light blue. The TALEN binding sites (for AGAP011377) are shown as underlined text.

Accession codes

Primary accessions

NCBI Reference Sequence

References

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Author information

Affiliations

  1. Department of Life Sciences, Imperial College London, London, UK.

    • Andrew Hammond,
    • Roberto Galizi,
    • Kyros Kyrou,
    • Alekos Simoni,
    • Dimitris Katsanos,
    • Matthew Gribble,
    • Austin Burt,
    • Nikolai Windbichler,
    • Andrea Crisanti &
    • Tony Nolan
  2. Dipartimento di Medicina Sperimentale Via Gambuli, Centro di Genomica Funzionale, University of Perugia, Perugia, Italy.

    • Carla Siniscalchi
  3. Department of Genetics, University of Cambridge, Cambridge, UK.

    • Dean Baker &
    • Steven Russell
  4. INSERM U963, CNRS UPR9022, Université de Strasbourg, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France.

    • Eric Marois

Contributions

A.H., R.G., K.K., A.S., C.S., D.K. and M.G. performed the experiments. E.M. and N.W. developed mosquito reagents. D.B., S.R., T.N. and A.B. developed the logistic regression model. A.B. did the experimental modeling. A.H., R.G., N.W. and T.N. designed the experiments. A.H., A.C., A.B. and T.N. analyzed the data. A.H., A.C. and T.N. wrote the paper.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Molecular confirmation ofCRISPR–induced HDR at 3 candidate female fertility loci (142 KB)

    A PCR was performed using a primer pair (blue arrows) specific for either the 5′ or 3′ side of the putative integration of the hdrGFP docking construct. In each case a primer binding internal to the hdrGFP construct was combined with a primer binding external to the region of homology included in the hdrGFP plasmid such that an amplicon of the expected size should only be produced in the event of the precise homol-ogy-dependent repair event illustrated. 3 individuals of each line were tested.

  2. Supplementary Figure 2: Docking lines containing HDR gene disruptions show sterility at different stages of egg maturation. (88 KB)

    Females homozygous and heterozygous for the hdrGFP allele were mated in groups to wild type males and al-lowed to lay individually and the number of resulting eggs and larvae counted. A minimum of 20 individuals were tested for each line.

  3. Supplementary Figure 3: Molecular confirmation of CRISPRh alleles at candidate female fertility loci (168 KB)

    Following RMCE with a CRISPRh construct a PCR was performed to confirm the molecular nature of the event using a primer pair (blue arrows) designed to amplify from the docking site in the respective gene (blue bar) to an internal region of the CRISPRh construct. Primer pairs were designed for both the 5′ and 3′ ends of the integration. Nature Biotechnology

  4. Supplementary Figure 4: NHEJ and non–canonical CRISPR induced homing events at target loci (201 KB)

    32 progeny from CRISPRh/+ parents that did not show the RFP+ phenotype associated with the CRISPRh con-struct were analysed. The sequence included in the protospacer of the guide RNA is highlighted, with the PAM region in blue. Across the 3 loci we recovered 15 instances of mutations at the target loci and 17 instances showing wild type sequences. Among the mutations we recovered, 6 unique (originating from separate parents) NHEJ events resulting in indels and one unique incomplete homing event at AGAP007280 that incorporated a small region surrounding the guide RNA in the homing construct (lower panel).

  5. Supplementary Figure 5: Somatic activity of vasa::Cas9 (302 KB)

    Progeny from a homozygous vasa::Cas9;3XP3::YFP male crossed to a heterozygous U6::gRNA(YFP) female show variegated YFP expression in somatic tissues, including eye, dorsal ganglion and anal palps (left) as well as extensive mottling throughout the body (right). The two phenotypes segregated approximately 50:50, consis-tent with somatic expression of vasa::Cas9 in all individuals and Mendelian segregation of the gRNA allele to half the progeny. Larvae were visualised under a GFP filter.

  6. Supplementary Figure 6: Schematic of the three target genes used in this study and the nuclease binding sequences. (184 KB)

    The CRISPR target sequences are highlighted in grey; each includes a protospacer adjacent motif (PAM) se-quence at the target locus (5′-NGG), highlighted in light blue. The TALEN binding sites (for AGAP011377) are shown as underlined text.

PDF files

  1. Supplementary Text and Figures (3,085 KB)

    Supplementary Figures 1–6

Excel files

  1. Supplementary Table 1 (11 KB)

    CRISPR or TALEN-mediated HDR to disrupt candidate female fertility genes and produce docking lines

  2. Supplementary Table 2 (10 KB)

    Sterility annotations and controlled vocabulary (FlyBase 2011_7)

  3. Supplementary Table 3 (10 KB)

    Logistic regression coefficients

  4. Supplementary Table 4 (10 KB)

    P(sterile) of target genes

  5. Supplementary Table 5 (17 KB)

    List of genes with a p(sterile) score of ≥0.5

Additional data