PROJECT SUMMARY There are >20,000 genes in the genome and though there has been progress in assigning many gene functions, we still do not know the function of numerous genes or their specific roles in affecting disease. Genes can direct diverse phenotypes in different cell types, cell states, or tissues, which makes gene annotation a staggering task. Studies are needed to determine each gene’s function in different contexts, but the scope of the problem is a challenge – it is necessary to assess many possible functions of 100s of genes and within the context of a complex cellular and extracellular milieu whose spatial architecture is an essential component of system behavior. In addition, some genes work coordinately or redundantly with other genes, making phenotypic annotation more challenging. Establishing the role of every gene in different normal and disease states would have an incalculable impact on biology and medicine. It would elicit novel intrinsic components of different diseases and thus facilitate the development of drugs to treat some of these diseases. The objective of this project is to establish a first-of-its-kind platform for spatial functional genomics (Perturb- map), which will enable 100s of genetic perturbations, including multi-gene knockouts, to be generated in parallel in tissues and tumors, and the effect of each perturbation on multiple intrinsic and extrinsic biological processes to be revealed at cellular, subcellular, and tissue level resolution. To reach our objective, we will develop novel technologies, methods, and software tools that will permit 100s of CRISPRs, along with dozens of biological phenotypes, to be spatially resolved within a tissue by high-dimensional imaging. To establish the potential of Perturb-map to address urgent unmet needs, we will apply the platform to one of the most pressing questions in immune oncology, namely how tumors prevent immune infiltration and subvert immunity. We will use Perturb- map to identify factors that regulate immune recruitment and exclusion from the tumor microenvironment and uncover genes controlling tumor resistance to immune clearance. The outcome of this project will be a transformative technology for functional genomics with spatial cellular and sub-cellular resolution and high-dimensional phenotyping. This would have unmatched capabilities to answer numerous questions in a broad array of biological areas, and to investigate entire classes of genes and phenotypes that could not be studied with existing functional genomics approaches; enabling highly scaled studies to identify genes that control processes such as: tissue and tumor organization, cell migration, invasion and metastasis, cell-cell interactions, and local immune cell recruitment and crosstalk. By being able to interrogate multiple gene perturbations within the same cell, Perturb-map will also enable genetic redundancies and synthetic lethal relationships to be identified, a major area of interest for development of targeted cancer therapies. Thus, the platform will have broad utility in numerous areas of basic and translational biology.

PROJECT NARRATIVE This project will establish a first-of-its-kind platform for spatial functional genomics (Perturb-map), which will enable broad phenotypic analysis of 100s of genes in parallel within a tissue or tumor at cellular resolution and with spatial architecture preserved. This will greatly accelerate functional annotation of the genome and discovery of tissue and tumor biological control in a manner and scale not currently possible, and thus transform efforts to uncover the intrinsic components of different diseases and facilitate the development of drugs to treat these diseases.