Anopheles gambiae is a mosquito that can infect humans with malaria. The doublesex gene encodes two genetic sequences, dsx-female and dsx-male that control whether a mosquito is a male or female. The female sequence contains a coding sequence called exon 5, which is highly conserved in Anopheles. In this study, the researchers found that disruption of exon 5 aimed at blocking the formation of dsx-female did not affect male development or fertility but caused females to have an intersex phenotype and be sterile. A CRISPR-Cas9 gene drive targeting this sequence spread rapidly in caged mosquitoes, and reached 100% prevalence in 7-11 generations. This caused egg production to decrease to the point of population collapse.
The researchers wanted to find a gene to disrupt using CRISPR-Cas9 in Anopheles that could be used in a gene drive to collapse a lab population of the mosquito.
CRISPR-Cas9 has been used in previous studies to create gene drives in Anopheles gambiae and Anopheles stephensi with the purpose of vector control. These studies focused on suppressing the reproductive capability of mosquito populations. According to mathematical modeling, this suppression can be achieved using gene drives targeting genes related to female fertility, or introducing interruptions on the Y chromosome (called a Y-drive). Both strategies cause decreases in the number of fertile females that would eventually cause population collapse.
However, these strategies have technical and scientific issues. Y-drives prove difficult because the transcription of sex chromosomes shut down during meiosis, making it difficult to pass on interruptions to the next generation. A gene drive used to disrupt fertility gene AGAP007280 initially spread through a population, but nuclease-resistant variants emerged and blocked the spread of the drive. Gene drive targets with functional or structural constraints that can prevent the development of nuclease resistance could create successful gene drives. The researchers evaluated the potential for disrupting sex determination in A. gambiae to block the formation of doublesex in females.
doublesex and sex differentiation in A. gambiae
In A. gambiae, doublesex (dsx) is alternatively spliced during transcription to produce male and female transcripts (AgdsxM and AgdsxF respectively). To understand whether dsx was a good target for a gene drive suppressing reproduction, the researchers disrupted a specific portion of dsx to prevent the formation of AgdsxF while leaving AgdsxM unaffected. To do so, they injected A. gambiae embryos with Cas9 and a single-guide RNA that was designed to recognize and cleave the interruption site, while an eGFP transcription was inserted into the site using a template for homology-directed repair. Transformed mosquitos were crossed to breed homozygous and heterozygous mutants. Larvae that were heterozygous for the disruption developed normally into male and female mosquitoes. However, half of the homozygous mutants had both male and female features, and abnormalities in reproductive organs (intersex). Intersex females were unable to take blood meals and failed to produce eggs.
Building a gene drive to target dsx
The researchers replaced the eGFP transcription insert with a dsxFCRISPRh gene drive construct so that the interruption could be passed onto future generations. The dsxFCRISPRh construct was able to bypass traditional inheritance and were passed on to 95.9% of heterozygous males and 99.4% of heterozygous females. dsxFCRISPRh heterozygous males had no change in fertility, while dsxFCRISPRh heterozygous females showed decreased fertility.
Assessment of dsx gene drive in caged insects
Using a mathematical model, the researchers calculated that dsxFCRISPRh could reach 100% prevalence in a mosquito population in 9-13 generations. To test this, the researchers mixed wild-type mosquitos with heterozygous dsxFCRISPRh mosquitos and monitored their progeny at each generation. They performed this in two cages. During the first 3 generations, the drive allele increased from 25% to ~69% in both cages. In one of the cages, the drive reached 100% frequency by generation 7 and population collapse was achieved at generation 8. In the other cage, 100% frequency was reached at generation 11, with population collapse at generation 12. Both of these results fell within the range of the mathematical model.
Potential for resistance to dsx gene drive
There was a low frequency of mutations in this study’s gene drive. There was no evidence of positive selection, which would have caused the drive to fail. The researchers found that the guide RNA used in the gene drive construct efficiently cleaved variants of dsx, suggesting that the gene drive would be successful even in the presence of dsx mutants. However, the drive is not “resistance-proof.”
The development of a gene drive capable of collapsing mosquito populations to a level that cannot support malaria transmission is a long-sought goal. The gene drive used in this study, with the dsxFCRISPRh has features that make it suitable for field testing. The drive has high inheritance rates, fully fertile heterozygous individuals, sterile homozygous females, and no evidence of nuclease resistance. Nonetheless, the drive is not resistance-proof.
The gene drive used in this study now needs to be evaluated in large confined spaces that mimic natural ecological conditions.