CNOT1 CRISPR Activation Plasmid (h): sc-403431-ACT

Human CNOT1 encodes a central scaffold of the CCR4–NOT complex, a multi-subunit regulator of gene expression that couples transcriptional control with mRNA deadenylation, decapping, and decay. By coordinating recruitment and activity of deadenylases and interacting with RNA-binding proteins and microRNA effector machinery, CNOT1 helps set transcript stability and translation efficiency across diverse cellular programs. This regulation is integral to cell-cycle progression, differentiation, stress responses, and maintenance of proteostasis through broad effects on mRNA turnover. Dysregulation of CCR4–NOT components, including CNOT1, has been associated with altered RNA homeostasis and transcriptional programs observed in cancer biology and neurodevelopmental phenotypes, supporting its relevance in mechanistic studies of disease-linked gene regulatory networks.

CNOT1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous CNOT1 expression without altering the underlying DNA sequence.

CNOT1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the CNOT1 locus in human cell lines. The system is built around a catalytically inactive Cas9 (dCas9) carrying two inactivating mutations (D10A and N863A) that eliminate nuclease activity while preserving DNA binding. This dCas9 is fused to VP64, a potent transcriptional activator, and is co-expressed with a blasticidin resistance gene for selection. The second plasmid encodes the MS2-p65-HSF1 fusion protein, a secondary activator complex that works in concert with dCas9-VP64, alongside a hygromycin resistance gene. The third plasmid encodes a target-specific 20 nt sgRNA fused to two MS2 RNA aptamers that recruit the MS2-p65-HSF1 complex to the activation site, accompanied by a puromycin resistance gene. The three plasmids are delivered at a 1:1:1 mass ratio for balanced expression of all system components.

Once assembled at the target locus, the SAM complex binds within approximately 200 bp upstream of the CNOT1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous CNOT1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native CNOT1 locus and enabling the study of CNOT1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of CNOT1 pathway restoration in tumor cells with silenced or reduced CNOT1 expression.

For Research Use Only. Not Intended for Diagnostic or Therapeutic Use.