The haplolethal gene wupA of Drosophila exhibits potential as a target for an X-poisoning gene drive.

A synthetic gene drive that targets haplolethal genes on the X chromosome can skew the sex ratio toward males. Like an "X-shredder," it does not involve "homing," and that has advantages including the reduction of gene drive resistance allele formation. We examine this "X-poisoning" strategy by targeting 4 of the 11 known X-linked haplolethal/haplosterile genes of Drosophila melanogaster with CRISPR/Cas9. We find that targeting the wupA gene during spermatogenesis skews the sex ratio so fewer than 14% of progeny are daughters. That is unless we cross the mutagenic males to X^XY female flies that bear attached-X chromosomes, which reverses the inheritance of the poisoned X chromosome so that sons inherit it from their father, in which case only 2% of the progeny are sons. These sex ratio biases suggest that most of the CRISPR/Cas9 mutants we induced in the wupA gene are haplolethal but some are recessive lethal. The males generating wupA mutants do not suffer from reduced fertility; rather, the haplolethal mutants arrest development in the late stages of embryogenesis well after fertilized eggs have been laid. This provides a distinct advantage over genetic manipulation strategies involving sterility which can be countered by the remating of females. We also find that wupA mutants that destroy the nuclear localization signal of shorter isoforms are not haplolethal as long as the open reading frame remains intact. Like D. melanogaster, wupA orthologs of Drosophila suzukii and Anopheles mosquitos are found on X chromosomes making wupA a viable X-poisoning target in multiple species.

Acetylcholine esterase of Drosophila melanogaster: a laboratory model to explore insecticide susceptibility gene drives.

BACKGROUND: One of the proposed applications of gene drives has been to revert pesticide resistant mutations back to the ancestral susceptible state. Insecticides that have become ineffective because of the rise of resistance could have reinvigorated utility and be used to suppress pest populations again, perhaps at lower application doses. RESULTS: We have created a laboratory model for susceptibility gene drives that replaces field-selected resistant variants of the acetylcholine esterase (Ace) locus of Drosophila melanogaster with ancestral susceptible variants. We constructed a CRISPR/Cas9 homing drive and found that homing occurred in many genetic backgrounds with varying efficiencies. While the drive itself could not be homozygous, it converted resistant alleles into susceptible ones and produced recessive lethal alleles that could suppress populations. Our studies provided evidence for two distinct classes of gene drive resistance (GDR): rather than being mediated by the conventional non-homologous end-joining (NHEJ) pathway, one seemed to involve short homologous repair and the other was defined by genetic background. Additionally, we used simulations to explore a distinct application of susceptibility drives; the use of chemicals to prevent the spread of synthetic gene drives into protected areas. CONCLUSIONS: Insecticide susceptibility gene drives could be useful tools to control pest insects however problems with particularities of target loci and GDR will need to be overcome for them to be effective. Furthermore, realistic patterns of pest dispersal and high insecticide exposure rates would be required if susceptibility were to be useful as a 'safety-switch' to prevent the unwanted spread of gene drives. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.