To uncover the human protein network underlying infection and to identify targets for novel host-directed anti‐infectives, InfectX (2009‐2013) employed RNA interference (RNAi) screens. In that project, libraries of small inhibitory RNAs (siRNAs) that systematically target a large fraction of human genes were screened, and the effects of individual siRNA perturbations on the cellular infection processes by various human pathogens were measured. The image‐based high‐content RNAi datasets that were generated through a standardized experimental and computational approach are of unprecedented quality and breadth and should thus enable systems‐level analyses of general gene‐phenotype relationships. SiRNAs are designed to be perfectly complementary to 19‐ 23 nucleotide regions in the intended mRNA targets. As siRNA targets undergo degradation upon siRNA transfection, it is generally assumed that the observed phenotypes are due to the direct effect of the siRNA on these targets (‘on‐target effect’). However, our systematic exploration of the impact of siRNA‐based RNAi on two phenotypic readouts (cell number and infection index) in InfectX has highlighted the prevalence and magnitude of ‘off‐target effects’ that are mediated by the siRNA ‘seed sequences’ (nucleotides 2‐8 from the 5’ end of the siRNA) through a microRNA (miRNA)‐type mechanism. A major contribution that InfectX made to the RNAi screening field was the development of modeling approaches to comprehensively quantify off‐target effects, partly deconvolute combinatorial effects, and finally provide improved methods for establishing individual gene‐phenotype relationships. The present proposal, TargetInfectX (2014‐2017), will build on the image‐based high‐content RNAi datasets established within InfectX, but will have more far‐reaching goals. From the image data generated in InfectX we will extract a rich set of phenotypic features on the single‐cell level. Modeling miRNAtarget mRNA interactions, their effect on gene expression, and the gene‐phenotype relationships emerging from the imaging data, we will attempt to generally reconstruct the genotypic basis of elementary cell behaviors such as those involved in the response to pathogen intrusion. On the translational side, we will explore the potential of siRNAs as novel combination therapy approach to interfere with the course of infections. Moreover, beyond the public release of our data via publications and publically accessible databases, we will encourage knowledge and technology transfer of unpublished work in progress with partners outside of TargetInfectX via a collaboration suite that should foster long‐term collaborations and ensure the impact of our work beyond SystemsX.ch.