PROJECT SUMMARY In sexually reproducing species males and females have divergent morphologies, behaviors, and reproductive strategies to reach their respective fitness optima while sharing a largely similar genome. These differences can cause sexual conflict, wherein the presence of a particular trait can result in opposite fitness effects in males and females. In intralocus sexual conflict (ISC) the presence of a single gene within a locus can increase the fitness of one sex while decreasing the fitness of the other. There is widespread evidence of ISC occurring in many animals, including humans, and the presence of sexual dimorphism suggests that some aspects of ISC can be resolved. While previous theoretical and gene association studies have helped predict ISC and strategies for its mitigation, we still have little empirical evidence of which genes are involved in ISC, their roles, and their direct impacts on sex-specific fitness in evolution. Recently, our laboratory published the first direct evidence implicating new gene evolution through gene duplication in the resolution of ISC. We found that a pair of recently duplicated genes in Drosophila had quickly accumulated sequence changes and acquired essential, sex-specific functions in male and female reproduction—supporting previous theoretical predictions. This was a groundbreaking finding as it suggests that ISC resolution can drive new gene evolution. However, the generality of this single case study remains unknown. The objective of this proposal is to define the generality of sexual conflict-driven new gene evolution. I hypothesize that intralocus sexual conflict resolution can drive the evolution of new genes through rapid acquisition of novel functions which contribute to sex-specific fitness. I will test my hypothesis in two major Aims. First, I will use a CRISPR-Cas9-aided reverse genetic approach to assess the genetic necessity of 36 newly duplicated genes through sex-specific fitness assays. Thus far I have knocked out seven new genes and found that loss-of-function mutations in three genes reduced the fertility of male or female flies, suggesting they may have contributed to the resolution of an ancestral sexual conflict. Second, I will conduct a deeper functional analysis on five genes examined in Aim 1, using RNA sequencing at both the tissue and single cell levels, to identify pathways and cell types that require new gene functions in resolving sexual conflict. My proposed research combines a CRISPR-Cas9-based reverse genetics approach with transcriptome profiling to define the generality of ISC-driven new gene evolution and how these new genes facilitate sex-specific functions to mediate or resolve sexual conflict.