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.