UCSF

The flow of genetic information through transcription and translation—known as the central dogma of biology—enables all aspects of life, from the response of a solitary yeast cell to an environmental stimulus, to the intricate choreography guiding development of a single-celled zygote into a complex organism with functionally distinct tissues. Recent technological advances have provided tools to observe and/or perturb molecular processes underlying the central dogma with unprecedented resolution and precision. In this dissertation, I describe two cases where such tools were used to study and manipulate gene expression in eukaryotic cells.

First, I show that in budding yeast, under nutrient limiting conditions, the commonly used translation inhibitor cycloheximide induces rapid transcriptional upregulation of hundreds of genes involved in ribosome biogenesis. This generates a large pool of mRNAs that cannot be translated due to the presence of the inhibitor, leading to the appearance of strong translational regulation. This work provides a novel mechanistic interpretation for perplexing reports in the translation field, and hopefully serves to guide experimental design moving forward.

Second, I describe the development of allelic series of systematically compromised sgRNAs to titrate expression of human genes with CRISPR interference. Large-scale measurements of compromised sgRNA activities enable identification of empirically validated intermediate-activity sgRNAs and determination of the factors governing sgRNA activity using deep learning, facilitating construction of a compact sgRNA library to titrate expression of ~2,400 essential genes and a genome-wide in silico library. Staging cells along a continuum of essential gene expression levels using sgRNA series combined with rich single-cell RNA-seq readout reveals expression threshold-specific responses and gene-specific expression-to-phenotype relationships, thus highlighting such reagents as a general tool to titrate the expression of human genes, with potential applications ranging from tuning of biochemical pathways to identification of suppressors for diseases of dysregulated gene expression.