CRISPR activation screens for functional genomics discovery

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) activation (CRISPRa) has emerged as a powerful tool in molecular biology, enabling high-throughput identification of genes whose upregulation drives specific phenotypes. This thesis explores the versatility of CRISPRa genetic screens by integrating recent advances in CRISPRa technology with a strategic approach that combines parallel and complementary screening methods. By conducting multiple screens with distinct objectives, this work highlights the advantages of diverse screening perspectives to improve our understanding of methodologies and outcomes. The primary objectives were to identify novel receptors for angiotensin II and bradykinin, uncover genetic modulators of cell survival under drug-induced stress, and discover genes that enhance lipid nanoparticle (LNP) efficacy. These aims were pursued through a series of CRISPRa screens. Key findings include the identification and validation of previously unrecognized modulators of ligand binding, protective factors against drug toxicity, and enhancers of LNP-mediated mRNA expression. This study demonstrates that performing multiple CRISPRa screens in parallel not only strengthens dataset robustness but also offers opportunities to refine screening approaches and assess variability. These findings underscore the value of multifaceted screens in receptor discovery, drug-response pathway analysis, and LNP efficacy optimization. By broadening both scope and depth, this research highlights CRISPRa’s potential to address complex biological questions and drive innovation in molecular biology.