Gene Editing In A. Gambiae Mosquitos

Malaria is a deadly disease transmitted to humans by female mosquitos carrying the Plasmodium parasite. It is estimated that there was 216 million cases of malaria and as many as 445, 000 malaria deaths in 2016. A recent BBC article “Gene editing wipes out mosquitoes in the lab” discussed altering part of a gene called doublesex (dsx) to block reproduction in female mosquitos and ultimately collapse the population. Sex differentiation in A. gambiae mosquitos is controlled by alternative splicing in the dsx gene to produce the male and female transcripts (AgdsxM and AgdsxF). The female transcript contains a female specific exon (exon 5) and the intron 4-exon 5 boundary was targeted with CRISPR-Cas9, a technique used to alter DNA, to prevent the formation of functional AgdsxF.

The gRNA sequence used to target the gene and that spans the intron 4-exon 5 boundary is shaded in grey. A. gambiae embryos were injected with Cas9 and a single-guide RNA made to recognise and split a sequence overlapping the intron 4-exon 5 boundary, along with a template for homology directed repair (HDR) to introduce an eGFP transcription unit. They were then intercrossed to produce homozygous and heterozygous mutants. The eGFP resulted in the exon 5 coding sequence being disrupted which was proved by PCR and genomic sequencing. Half of mosquitos homozygous for the dsxF- allele developed into normal males while the other half possessed abnormalities in their internal and external reproductive organs, displaying an intersex phenotype. To determine the sex genotype of the intersex mosquitos, the mutation was introgressed into a line with a Y linked marker (RFP) and the presence of this marker was used to appoint sex genotypes. This showed that the intersex phenotype was seen only in females homozygous for the null mutation. These mosquitos were incapable of taking a blood meal or laying eggs.

A gene drive is a system that greatly increases the chance of a gene being passed onto offspring. A gene drive to target dsx was built by using recombinase-mediated cassette exchange to replace the 3xP3::GFP transcription unit with a dsxFCRISPRh gene drive construct. While meiosis happens, the Cas9-gRNA complex splits the wildtype allele at the target sequence and the dsxFCRISPRh cassette is duplicated into the wildtype locus by HDR, breaking exon 5. Using a mathematical model it was found that the dsxFCRISPRh had the ability to reach 100% frequency in caged populations in between 9-13 generations provided a starting allele frequency of 12. 5% and stochasticity. To test this, wildtype mosquitos were mixed with mosquitos carrying the dsxFCRISPRh allele and monitored to assess the spread of the drive. In cage 1 the drive allele frequency reached 100% at generation 11, and in cage 2 reached 100% by generation 7. Drive allele reached 100% in cage 1 (red) at generation 11, and in cage 2 (blue) at generation 7. The black line represents a deterministic model while the grey lines are stochastic simulations.

Using a CRISPR-Cas9 gene drive to target dsx and suppress reproductive capability in the A. gambiae mosquito population has potential as shown by the population collapse in the experiment. It now needs to be trialled in spaces that resemble real life conditions.