Multiplexed, Continuous Reporting of Gene Expression via CRISPR-mediated Transcriptional Activation

PROJECT SUMMARY: Although the detection of gene expression by quantitative PCR, bulk or single cell RNA sequencing represents a cornerstone of biological inquiry, these approaches are limited in their ability to measure transcriptional dynamics over time and throughout the spatial heterogeneity of tissues, and to detect low-abundance transcripts including those from cytokines. Cytokine expression shapes the infiltration and activity of immune cells in tumors and tissues and represents an important correlate of response to immunotherapy. However, cytokine expression is context-dependent, pleiotropic, dynamic and asynchronous. As such, it is particularly poorly suited to single ‘snapshot’ measurements in time, or the detection of a single or even a few inflammatory mediators in isolation. To address these challenges, we will combine advances in CRISPR- mediated transcriptional activation technology with genetically-encoded set reporting, to fluorescently report the transcriptional activity of 9 or more cytokines simultaneously, dynamically and in situ in tissues. We have previously validated the core capability of our system to report single gene expression in preliminary studies and will now optimize its detection capabilities and implement a Cre-Lox-based randomized fluorescent reporter set-encoding strategy to multiplex detection. If successful, our study will establish a novel approach to multiplexed and high-sensitivity CRISPR-based detection of gene expression. It will also produce tools and reagents that can be directly applied to interrogate the basis for tumor microenvironment inflammation and can be adapted for functional genomic and in vivo studies.