Applications of forward genetic screens to LncRNAs, cancer immunotherapy, and cellular engineering

Abstract

Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. By perturbing thousands of genes simultaneously and selecting for a desired phenotype, genetic features can be systematically mapped to phenotypic changes. Recently, CRISPR-Cas9 has emerged as a powerful genetic perturbation technology, opening up new opportunities for forward genetic screens. In this thesis, I present work to advance this approach and demonstrate its application in a range of contexts relevant to human health. We first established a detailed CRISPR-Cas9 screening protocol that outlines experimental design considerations. We then applied this methodology to develop a CRISPR toolkit for screening and characterizing long non-coding RNAs in the human genome, many of which remain uncharacterized. We identified the EMICERI locus as a regulator of four neighboring genes, one of which conferred resistance to a melanoma therapeutic. We next sought to use CRISPR activation screening to gain insight into the cellular processes that govern tumor resistance to immunotherapy. We identified four candidate genes in our screen, which we validated in diverse cancer cell types and explored through mechanistic studies, leading to the discovery of novel immunotherapy resistance pathways. Finally, we developed a pooled transcription factor (TF) screening platform that provides a generalizable approach for studying cellular programming. We created a comprehensive human TF library and applied it to identify TFs that can drive differentiation of embryonic stem cells toward neural cell fates. We discovered that one TF, RFX4, leads to differentiation of neural progenitors that produced inhibitory neurons, providing an efficient method for generating this important cell type. During the COVID-19 pandemic, we paused the screening work and developed a streamlined SARS-CoV-2 detection assay, STOPCovid, suited for low-complexity settings. STOPCovid combines viral RNA concentration with isothermal amplification and CRISPR-mediated detection. STOPCovid achieved a sensitivity and specificity of 93.1% and 98.5%, respectively, on patient samples. Together, our applications of forward genetic screens address diverse problems in human health and broadly demonstrate the potential of this approach for systematically interrogating genetic elements.