Driving into doublesex

Shwetha Shivaprasad

Shwetha Shivaprasad, Ph.D. is a posdoctoral researcher at Stanford University where she studies the host proteins interacting with the mammalian and mosquito adapted variants of dengue.

Kyrou et al. (2018) in a recent paper in Nature Biotechnology report identifying a highly conserved target site in the Anopheles gambiae fertility gene doublesex (dsx), that cannot tolerate mutations. They built a gene drive targeting this site in dsx which caused the mosquito population to collapse in 7-11 generations without emergence of resistant mutants.

CRISPR-based gene drives are actively being explored for potential use in vector control strategies to limit the spread of mosquito borne diseases such as malaria, Dengue and Zika. They supersede Mendelian inheritance allowing a particular gene variant to quickly spread through a population. Gene drives targeting female fertility genes in Anopheles gambiae mosquitoes were built to suppress their reproduction and annihilate mosquito populations. However, they were unsuccessful due to the emergence of mutations in these fertility genes that make them resistant to cleavage by homing endonucleases.

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doublesex transcript splicing patterns in males and females of D. melanogaster and A. gambiae. Kyrou et al. targeted their gene drive element to the 3′ splice junction of intron 4. This region of the gene is highly constrained because of its essential role in the formation of fully functional dsx transcripts in females.

doublesex gene products are critical for sex determination in mosquitoes. There are two alternatively spliced transcripts, specific to males or females. The female dsx transcript has an extra exon 5, which the authors found is highly conserved among 16 different anopheline species. The high degree of sequence conservation indicated that dsx exon 5 is under tight functional constraint and may not be able to tolerate mutations without compromising gene function.

When dsx exon 5 was disrupted using CRISPR, it resulted in loss of female fertility in dsxF−/− homozygous female mosquitoes and they showed an intersex phenotype with morphological features of both males and females and developmental anomalies in reproductive organs. They were unable to feed on a blood meal or lay eggs. The absolute requirement of dsxF for fertility in female mosquitoes and its possible resistance to acquiring functional mutations made it an attractive gene drive target. The authors predicted that the dsxF exon 5 disruption could be propagated in a mosquito population using gene drive systems, progressively increasing the number of non-egg laying intersex females and eventually exterminating the mosquito population.

Image result for gene driveThey tested their hypothesis using a dsxFCRISPRh gene drive construct expressing the guide RNA targeting dsx exon5 and the Cas9 endonuclease under germline promoter zpg. As expected from a gene drive, the dsxFCRISPRh allele bypassed Mendelian inheritance and was transmitted to a larger number of larval progeny. In addition to the intersex phenotype shown by dsxFCRISPRh homozygous females, the dsxFCRISPRh/+ heterozygous females also showed a somatic mosaic phenotype and reduced fertility.

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Anopheles gambiae

When dsxFCRISPRh/+ male mosquitoes were released into cages of wildtype mosquitoes at a frequency of 12.5%, which is very similar to the numbers used in field release experiments, the dsxFCRIPSRh allele penetrated the entire caged mosquito population in 7-11 generations as predicted by the authors through mathematical modeling. Also, the dsxF gene did not accumulate any functional, nuclease resistant mutations which could potentially block the spread of the gene drive. Even a rare, naturally occurring SNP variant of dsxF could be successfully cleaved by the Cas9 endonuclease indicating that this gene is a good potential target for gene drive based vector control. But the success of any gene drive depends on multiple environmental factors and selection pressures that are hard to predict and need to evaluated in larger, more ecologically relevant conditions.




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