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.
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- 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.
- 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.
- 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
- 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).
- 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.
- 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.
- Supplementary Text and Figures (3,085 KB)
Supplementary Figures 1–6
- Supplementary Table 1 (11 KB)
CRISPR or TALEN-mediated HDR to disrupt candidate female fertility genes and produce docking lines
- Supplementary Table 2 (10 KB)
Sterility annotations and controlled vocabulary (FlyBase 2011_7)
- Supplementary Table 3 (10 KB)
Logistic regression coefficients
- Supplementary Table 4 (10 KB)
P(sterile) of target genes
- Supplementary Table 5 (17 KB)
List of genes with a p(sterile) score of ≥0.5