This study presents a robust technology for systematically investigating the cellular consequences of repressing or inducing individual transcripts using CRISPRi and CRISPRa. The authors developed a method to modulate transcription of endogenous genes in human cells using catalytically inactive Cas9 (dCas9) fusion proteins guided by gene-specific sgRNAs. They performed a high-throughput tiling screen to define rules for CRISPRi activity at endogenous genes, identifying optimal sgRNA sequences and windows for targeting. This information was used to construct genome-scale CRISPRi and CRISPRa libraries, which were validated through growth-based screens and a toxin sensitivity screen. The results demonstrate that CRISPRi and CRISPRa can be used to modulate gene expression over a wide range, identify essential genes, tumor suppressors, and regulators of differentiation, and provide insights into the mechanisms of pathogen entry and toxicity. The study highlights the potential of CRISPRi and CRISPRa as powerful tools for mapping complex pathways and understanding transcript function.This study presents a robust technology for systematically investigating the cellular consequences of repressing or inducing individual transcripts using CRISPRi and CRISPRa. The authors developed a method to modulate transcription of endogenous genes in human cells using catalytically inactive Cas9 (dCas9) fusion proteins guided by gene-specific sgRNAs. They performed a high-throughput tiling screen to define rules for CRISPRi activity at endogenous genes, identifying optimal sgRNA sequences and windows for targeting. This information was used to construct genome-scale CRISPRi and CRISPRa libraries, which were validated through growth-based screens and a toxin sensitivity screen. The results demonstrate that CRISPRi and CRISPRa can be used to modulate gene expression over a wide range, identify essential genes, tumor suppressors, and regulators of differentiation, and provide insights into the mechanisms of pathogen entry and toxicity. The study highlights the potential of CRISPRi and CRISPRa as powerful tools for mapping complex pathways and understanding transcript function.