A CRISPR-Cas9-based transcriptional activation system was developed to specifically activate endogenous human genes using RNA-guided transcription factors. This system involves fusing dCas9 (a Cas9 variant with no endonuclease activity) to a transactivation domain (VP64) and targeting it to DNA sequences via guide RNAs (gRNAs). The study demonstrates that co-expression of this transactivator with gRNAs can induce specific expression of endogenous target genes in human cells, offering a simple and versatile approach for RNA-guided gene activation. The system was tested by targeting the promoter of the IL1RN gene with four gRNAs, resulting in significant activation of IL1RN expression. The study also confirmed that the dCas9-VP64 system does not cause DNA cleavage at target sites, as evidenced by the absence of DNA repair events. Additionally, the system was shown to activate the expression of other genes, including ASCL1, NANOG, HBG1/2, MYOD1, VEGFA, TERT, IL1B, and IL1R2, when targeted to their respective promoters. The study highlights the potential of this system for a wide range of applications in cell and gene therapy, genetic reprogramming, and regenerative medicine. It also demonstrates that the system can activate gene expression in other cell types, such as murine embryonic fibroblasts (MEFs), when targeting the ASCL1 promoter. The system was found to be highly specific, with no detectable off-target effects, as shown by RNA-seq analysis. The study also discusses the advantages of this system, including its potential for rapid and economical generation of new gene activators through simple synthesis of gRNA expression plasmids. The system is also compatible with direct in vitro transcription and transfection, making it a versatile tool for gene regulation. However, the study notes that the activation of IL1RN by dCas9-VP64 was weaker compared to TALE-based transcription factors, and further studies are needed to understand the differences between these technologies. Overall, the study presents a new and effective method for activating endogenous mammalian genes using RNA-guided transcription factors, offering a promising approach for targeted gene regulation.A CRISPR-Cas9-based transcriptional activation system was developed to specifically activate endogenous human genes using RNA-guided transcription factors. This system involves fusing dCas9 (a Cas9 variant with no endonuclease activity) to a transactivation domain (VP64) and targeting it to DNA sequences via guide RNAs (gRNAs). The study demonstrates that co-expression of this transactivator with gRNAs can induce specific expression of endogenous target genes in human cells, offering a simple and versatile approach for RNA-guided gene activation. The system was tested by targeting the promoter of the IL1RN gene with four gRNAs, resulting in significant activation of IL1RN expression. The study also confirmed that the dCas9-VP64 system does not cause DNA cleavage at target sites, as evidenced by the absence of DNA repair events. Additionally, the system was shown to activate the expression of other genes, including ASCL1, NANOG, HBG1/2, MYOD1, VEGFA, TERT, IL1B, and IL1R2, when targeted to their respective promoters. The study highlights the potential of this system for a wide range of applications in cell and gene therapy, genetic reprogramming, and regenerative medicine. It also demonstrates that the system can activate gene expression in other cell types, such as murine embryonic fibroblasts (MEFs), when targeting the ASCL1 promoter. The system was found to be highly specific, with no detectable off-target effects, as shown by RNA-seq analysis. The study also discusses the advantages of this system, including its potential for rapid and economical generation of new gene activators through simple synthesis of gRNA expression plasmids. The system is also compatible with direct in vitro transcription and transfection, making it a versatile tool for gene regulation. However, the study notes that the activation of IL1RN by dCas9-VP64 was weaker compared to TALE-based transcription factors, and further studies are needed to understand the differences between these technologies. Overall, the study presents a new and effective method for activating endogenous mammalian genes using RNA-guided transcription factors, offering a promising approach for targeted gene regulation.