Project Summary/Abstract. Antibodies are an invaluable tool in the biological sciences and are increasingly used as therapeutics. Over the past few years a large number of proteomic techniques and large scale studies have been developed to probe biological and biochemical processes at ever increasing scales. Many of these methods have been limited by the availability of quality antibody reagents which are still generated one-by-one for each antigen target and the lack of antibody sequences available. Additionally, large concerns have been raised regarding the quality and reproducibility of current commercial antibodies. While high-throughput methods have been described capable of generating antibodies against hundreds of antigens, they are slow, laborious, usually require significant automation, and are dependent on antigen availability. These issues, combined with the growing need for many quality antibodies, highlight the urgent need to develop methods capable of proteome-scale antibody generation. Here I introduce a radical proposal to create a platform which can generate antibodies against all potential antigen targets in a target proteome and subsequently carry out proteome-scale tests of cross-reactivity. This approach will be demonstrated on the human and zebrafish proteomes and consists of three parts. First, I will develop gene synthesis technologies to create proteome-scale gene libraries consisting of tens of thousands of antigens. Second, I will generate high diversity libraries of antibodies by repurposing diversity generating retroelements to carry out targeted mutagenesis in-vivo. This will allow us to bypass the diversity bottlenecks normally imposed by transformation. Third, I will carry out a large scale library-on-library selection for antibody-antigen interactions in-vivo using a multiplexed protein fragment complementation assay. The resulting antibody hits will be rescreened against all antigens to measure their cross-reactivity. This study will reduce the cost of antibody generation by at least two-orders of magnitude and lead to the rapid creation of large libraries of high-specificity, sequence-verified antibodies which, given the ubiquitous nature of these reagents, will have a significant impact on all areas of basic and clinical research. Furthermore, the technological developments in gene-synthesis and targeted in-vivo mutagenesis outlined will here have broad applicability in many areas of life sciences research.