UC Berkeley

Genome-wide CRISPR screens have emerged as powerful tools for uncovering the genetic underpinnings of diverse biological processes. Incisive screens often depend on directly measuring molecular phenotypes, such as regulated gene expression changes, provoked by CRISPR-mediated genetic perturbations. Here, we provide quantitative measurements of transcriptional, post-transcriptional, and translational responses in human cells across genome-scale perturbation libraries by coupling CRISPR interference (CRISPRi) with barcoded expression reporter sequencing. This approach, CiBER-seq, uses bulk RNA sequencing alone to read out reporter activity across complex perturbation libraries. In Chapter 2, we develop and validate CiBER-seq in mammalian cells, optimizing the integration of highly complex, barcoded sgRNA libraries into a defined genomic context. CiBER-seq profiling of a nuclear factor kappa B (NF-κB) reporter delineates the canonical signaling cascade linking the transmembrane TNF-α receptor to inflammatory gene activation while also identifying cell-type-specific regulators. In Chapter 3, we apply CiBER-seq to characterize the time-, dose-, and condition-dependent dynamics of the mammalian unfolded protein response, recovering known pathway components and revealing context-specific genetic dependencies. In Chapter 4, we use CiBER-seq to investigate regulators of mRNA export, a phenotype uniquely accessible to sequencing-based RNA reporter measurements at genome-scale. Finally, in Chapter 5, we extend CiBER-seq to translational reporters, enabling systematic dissection of translational regulation. Together, this work demonstrates the accuracy, versatility, and broad potential of CiBER-seq for mapping regulatory circuitry in mammalian cells.