A CRISPR/Cas9-based acetyltransferase, dCas9 fused to the p300 catalytic domain, was developed to activate genes from promoters and enhancers through targeted epigenetic modification. This fusion protein catalyzes histone H3K27 acetylation at target sites, leading to robust transcriptional activation of genes. The p300 domain is highly specific and can be ported to other programmable DNA-binding proteins, enabling versatile gene regulation. The system demonstrated strong activation of genes from both proximal and distal enhancers, with higher efficiency compared to conventional dCas9-based activators. The dCas9p300 Core fusion protein showed high specificity and robust gene activation, with minimal off-target effects. It also effectively acetylated H3K27 at enhancer and promoter regions, indicating its role in enhancer activity. The system can be used with a single guide RNA for gene activation, making it efficient for multiplexing multiple targets. The p300 Core domain was shown to be compatible with various DNA-binding proteins, including TALEs and zinc finger proteins, expanding its utility. The study highlights the potential of this system for precise epigenetic editing and gene regulation, with applications in gene therapy, regenerative medicine, and drug screening. The results support the use of targeted acetylation as a causal mechanism for gene activation and demonstrate the effectiveness of the CRISPR/Cas9-based approach in epigenome editing.A CRISPR/Cas9-based acetyltransferase, dCas9 fused to the p300 catalytic domain, was developed to activate genes from promoters and enhancers through targeted epigenetic modification. This fusion protein catalyzes histone H3K27 acetylation at target sites, leading to robust transcriptional activation of genes. The p300 domain is highly specific and can be ported to other programmable DNA-binding proteins, enabling versatile gene regulation. The system demonstrated strong activation of genes from both proximal and distal enhancers, with higher efficiency compared to conventional dCas9-based activators. The dCas9p300 Core fusion protein showed high specificity and robust gene activation, with minimal off-target effects. It also effectively acetylated H3K27 at enhancer and promoter regions, indicating its role in enhancer activity. The system can be used with a single guide RNA for gene activation, making it efficient for multiplexing multiple targets. The p300 Core domain was shown to be compatible with various DNA-binding proteins, including TALEs and zinc finger proteins, expanding its utility. The study highlights the potential of this system for precise epigenetic editing and gene regulation, with applications in gene therapy, regenerative medicine, and drug screening. The results support the use of targeted acetylation as a causal mechanism for gene activation and demonstrate the effectiveness of the CRISPR/Cas9-based approach in epigenome editing.