The study presents a CRISPR-based system for precise and modular regulation of transcription in eukaryotic cells. The dCas9 protein, which is catalytically inactive, is fused to effector domains that can repress or activate gene expression. This system allows for the targeted regulation of gene expression in human and yeast cells using short guide RNAs (sgRNAs). The dCas9-KRAB fusion protein effectively represses gene expression, while the dCas9-VP64 and dCas9-p65AD fusion proteins activate gene expression. The system is highly specific, with minimal off-target effects, as demonstrated by RNA-seq analysis. The CRISPRi system can silence endogenous genes in human and yeast cells, and it can be used for multiplexed gene regulation. The study also shows that CRISPRi can be used to map and perturb regulatory elements, such as enhancers, in human cells. The system provides a general method for efficiently and specifically regulating transcription in many eukaryotic cells. The study highlights the potential of CRISPRi as an alternative to RNAi for gene regulation, offering a modular and programmable approach for targeting proteins to DNA sequences. The results demonstrate that CRISPRi is a powerful tool for gene regulation in eukaryotic cells.The study presents a CRISPR-based system for precise and modular regulation of transcription in eukaryotic cells. The dCas9 protein, which is catalytically inactive, is fused to effector domains that can repress or activate gene expression. This system allows for the targeted regulation of gene expression in human and yeast cells using short guide RNAs (sgRNAs). The dCas9-KRAB fusion protein effectively represses gene expression, while the dCas9-VP64 and dCas9-p65AD fusion proteins activate gene expression. The system is highly specific, with minimal off-target effects, as demonstrated by RNA-seq analysis. The CRISPRi system can silence endogenous genes in human and yeast cells, and it can be used for multiplexed gene regulation. The study also shows that CRISPRi can be used to map and perturb regulatory elements, such as enhancers, in human cells. The system provides a general method for efficiently and specifically regulating transcription in many eukaryotic cells. The study highlights the potential of CRISPRi as an alternative to RNAi for gene regulation, offering a modular and programmable approach for targeting proteins to DNA sequences. The results demonstrate that CRISPRi is a powerful tool for gene regulation in eukaryotic cells.