While the catalog of mammalian transcripts and their expression levels in different cell types and disease states is rapidly expanding, our understanding of transcript function lags behind. We present a robust technology enabling systematic investigation of the cellular consequences of repressing or inducing individual transcripts. We identify rules for specific targeting of transcriptional repressors (CRISPRi), typically achieving 90%–99% knockdown with minimal off-target effects, and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9. Together they enable modulation of gene expression over a ∼1,000-fold range. Using these rules, we construct genome-scale CRISPRi and CRISPRa libraries, each of which we validate with two pooled screens. Growth-based screens identify essential genes, tumor suppressors, and regulators of differentiation. Screens for sensitivity to a cholera-diphtheria toxin provide broad insights into the mechanisms of pathogen entry, retrotranslocation and toxicity. Our results establish CRISPRi and CRISPRa as powerful tools that provide rich and complementary information for mapping complex pathways. Display omitted •CRISPRi and CRISPRa provide complementary information for mapping complex pathways•CRISPRi/a expression series (up to ∼1,000-fold) reveal how gene dose controls function•CRISPRi provides strong (typically 90%–99%) knockdown with minimal off-target effects•Genome-scale screens elucidate pathways controlling cholera/diphtheria toxicity Genome-scale-specific targeting of transcriptional repressors (CRISPRi) and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9 have been applied to growth and toxin-resistance screens, establishing CRISPRi and CRISPRa as powerful tools that provide rich and complementary information.