ABSTRACT For decades, biologists have taken parts from disparate proteins and fused them in various combinations to create engineered variants with user defined properties. Despite the success of many of the generated tools (e.g. chimeric antigen receptors and enhanced CRISPR variants) the methods by which these proteins are discovered are slow and labor intensive, limiting our exploration to only a tiny fraction of potential protein space. Here, we introduce BArcoded Combinatorial Engineering and Screening (BaCES), a method that enables the simultaneous assembly and parallel testing of tens of thousands of combinatorial protein variants. The objective of this proposal is to use BaCES to create a new generation of enhanced Cas9-based transcriptional regulators, which will be combined with a novel experimental paradigm to probe gene function within in vivo contexts. The rationale underlying this proposal is that, if successful, we will create several transformative technologies and gain insight into the mechanism by which neurons tolerate neurodegenerative insults. Herein we demonstrate the feasibility of our BaCES platform and provide evidence supporting our unique approach to in vivo screening. To further our research goals, we will: 1) use BaCES to generate and quantify the behavior of 27,000 Cas9 activators and repressors; 2) thoroughly validate across targets and cell types a new generation of highly-potent Cas9 transcriptional modulators; and 3) apply these tools to perform a set of in vivo genetic screens to uncover regulators of neuronal survival within a mouse model of Parkinson’s Disease. This proposal is innovative from a technical perspective in that it creates a new method for rapidly searching through combinatorial protein space and implements a new paradigm for performing in vivo CRISPR screens within a complex cellular environment. It is also innovative in approach as it utilizes a high-throughput platform to gain insight into the genes and pathways that regulate neuronal survival within an in vivo model of disease. This work is significant in that it will create a novel method for performing combinatorial protein screens, identify a set of enhanced Cas9 activators and repressors to enable global research endeavors, and uncover the biological processes that neurons use to tolerate neurodegenerative disease- associated stressors. Our track record of producing widely adopted CRISPR tools, combined with our preliminary data demonstrating the feasibility of the proposed work and a group of long- standing committed collaborators, makes our team uniquely suited to carry out the outlined interdisciplinary research.