Chk1 CRISPR Activation Plasmid (h): sc-400223-ACT

CHEK1 encodes the serine/threonine kinase Chk1, a central effector of the DNA damage response that coordinates S-phase and G2/M checkpoint control. Activated primarily downstream of ATR in response to replication stress and single-strand DNA lesions, Chk1 phosphorylates targets that regulate origin firing, replication fork stability, and cell-cycle progression, including CDC25 phosphatases and components of the replication machinery. Through these pathways, CHEK1 helps preserve genome integrity and limit accumulation of chromosomal aberrations. Dysregulated Chk1 signaling is frequently studied in the context of proliferative disorders, oncogene-induced replication stress, and mechanisms of resistance to genotoxic perturbations.

Chk1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous CHEK1 expression without altering the underlying DNA sequence.

Chk1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the CHEK1 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 CHEK1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Chk1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native CHEK1 locus and enabling the study of Chk1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Chk1 pathway restoration in tumor cells with silenced or reduced CHEK1 expression.

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