A robust technology is presented for systematically investigating the cellular consequences of repressing or inducing individual transcripts. The study identifies rules for specific targeting of transcriptional repressors (CRISPRi) and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9, enabling modulation of gene expression over a 1000-fold range. Genome-scale CRISPRi and CRISPRa libraries were constructed and validated with two pooled screens. Growth-based screens identified essential genes, tumor suppressors, and regulators of differentiation. Screens for sensitivity to a cholera-diphtheria toxin provided insights into pathogen entry, retro-translocation, and toxicity. The results establish CRISPRi and CRISPRa as powerful tools for mapping complex pathways. CRISPRi can repress transcription by directly blocking RNA polymerase activity or through effector domain-mediated transcriptional silencing. CRISPRa activates transcription by recruiting multiple copies of the activating effector domain. The study shows that CRISPRi and CRISPRa can activate and repress the transcription of endogenous genes over a wide dynamic range. The results demonstrate that CRISPRi is highly specific and non-toxic. The study also shows that CRISPRa can be used to identify regulators of survival and differentiation in human cells. The results highlight the utility of CRISPRi and CRISPRa for future studies into tumor biology and cell differentiation. The study also shows that CRISPRi can effectively repress the transcription of long noncoding RNAs. The results demonstrate that CRISPRi and CRISPRa are robust tools for systematically manipulating transcription of endogenous genes in human cells. The study shows that CRISPRi and CRISPRa can be used to rapidly screen for both loss-of-function and gain-of-function phenotypes in a pooled format. The study also shows that CRISPRi and CRISPRa can be used to create allelic series of gene expression, spanning a broad range from 100-fold repression to 10-fold induction. The study highlights the capacity of CRISPRa to complement CRISPRi by querying the consequences of upregulating pathways that may otherwise be inactive. The study also shows that CRISPRi can effectively repress lncRNA expression, enabling future systematic studies of non-coding gene function. The study demonstrates that CRISPRi and CRISPRa are robust tools for systematically manipulating transcription of endogenous genes in human cells. The study shows that CRISPRi and CRISPRa can be used to rapidly screen for both loss-of-function and gain-of-function phenotypes in a pooled format. The study also shows that CRISPRi and CRISPRa can be used to create allelic series of gene expression, spanning a broad range from 100-fold repression to 10-fold induction. The study highlightsA robust technology is presented for systematically investigating the cellular consequences of repressing or inducing individual transcripts. The study identifies rules for specific targeting of transcriptional repressors (CRISPRi) and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9, enabling modulation of gene expression over a 1000-fold range. Genome-scale CRISPRi and CRISPRa libraries were constructed and validated with two pooled screens. Growth-based screens identified essential genes, tumor suppressors, and regulators of differentiation. Screens for sensitivity to a cholera-diphtheria toxin provided insights into pathogen entry, retro-translocation, and toxicity. The results establish CRISPRi and CRISPRa as powerful tools for mapping complex pathways. CRISPRi can repress transcription by directly blocking RNA polymerase activity or through effector domain-mediated transcriptional silencing. CRISPRa activates transcription by recruiting multiple copies of the activating effector domain. The study shows that CRISPRi and CRISPRa can activate and repress the transcription of endogenous genes over a wide dynamic range. The results demonstrate that CRISPRi is highly specific and non-toxic. The study also shows that CRISPRa can be used to identify regulators of survival and differentiation in human cells. The results highlight the utility of CRISPRi and CRISPRa for future studies into tumor biology and cell differentiation. The study also shows that CRISPRi can effectively repress the transcription of long noncoding RNAs. The results demonstrate that CRISPRi and CRISPRa are robust tools for systematically manipulating transcription of endogenous genes in human cells. The study shows that CRISPRi and CRISPRa can be used to rapidly screen for both loss-of-function and gain-of-function phenotypes in a pooled format. The study also shows that CRISPRi and CRISPRa can be used to create allelic series of gene expression, spanning a broad range from 100-fold repression to 10-fold induction. The study highlights the capacity of CRISPRa to complement CRISPRi by querying the consequences of upregulating pathways that may otherwise be inactive. The study also shows that CRISPRi can effectively repress lncRNA expression, enabling future systematic studies of non-coding gene function. The study demonstrates that CRISPRi and CRISPRa are robust tools for systematically manipulating transcription of endogenous genes in human cells. The study shows that CRISPRi and CRISPRa can be used to rapidly screen for both loss-of-function and gain-of-function phenotypes in a pooled format. The study also shows that CRISPRi and CRISPRa can be used to create allelic series of gene expression, spanning a broad range from 100-fold repression to 10-fold induction. The study highlights