Enabling in vivo barcoded single-cell multiomics-compatible genome-wide screens in personalized tumor models using defined-copy somatic transgenesis

PROJECT SUMMARY / ABSTRACT CRISPR technology have massively expanded our ability to interrogate the genetic mechanisms of cancer and define therapeutic avenues. Techniques have evolved over the past five years for the generation of massive scale CRISPR genetic screens linked to single cell transcriptomics. However, the ability to perform such screen in vivo in genetically engineered models remains cumbersome. Over the past several years, we have pioneered an electroporation-based somatic mutation method for rapid, non-invasive, somatic transgenesis for high throughput validation of tumor driver genes using mosaic analysis with dual recombinase-mediated cassette exchange (MADR). The central theme of this grant application is to generate, optimize, and validate MADR Perturb-seq—a novel suite of genetic tools that facilitate massive scale genetic screens in the context of both in vivo murine somatic transgenesis as well as inducible editing in human iPSC-derived organoids. We will exploit our experience with electroporation based modeling to rapidly optimize and empirically test MADR Perturb-seq. The overall objective of the proposal is to perform advanced development of this combined MADR-CRISPR approach to allow for generalized use in diverse tumor contexts and, therefore, demonstrate the potential of this technology to transform cancer research. We propose to carry out this work in three parts. The focus of Specific Aim 1 is to Optimize CRISPR elements, ORFs, and molecular barcodes for use with somatic transgenic patient-derived tumor signatures and validate genetic tools for enabling multiplex genetic screens in an off the shelf CRISPR/Cas9 mouse. The main goal of Specific Aim 2 is to validate MADR multiplexing screening in vivo at scale . Finally, in Aim 3, we will engineer approaches for highly multiplexed genetic screens using MADR combined with inducible elements and/or virally-delivered MADR elements in human engineered iPSCs. Successful completion of these experiments will yield unprecedented capabilities for genetically screening tumor mechanisms and potential therapeutic weaknesses.