Topo I CRISPR Activation Plasmid (h): sc-400965-ACT

Human TOP1 encodes DNA topoisomerase I (Topo I), a nuclear enzyme that resolves torsional stress generated during DNA replication and transcription by creating transient single-strand breaks and religating DNA. This activity supports DNA damage response signaling, chromatin dynamics, and maintenance of genome stability, linking TOP1 function to cell-cycle progression and transcriptional homeostasis. Dysregulated TOP1 expression or activity can alter replication stress tolerance and genome integrity, features frequently studied in cancer biology and in contexts of impaired DNA repair. TOP1-dependent processes are also relevant to investigations of transcription-associated genomic instability and cellular responses to topoisomerase poisons.

Topo I CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous TOP1 expression without altering the underlying DNA sequence.

Topo I CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the TOP1 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 TOP1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Topo I expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native TOP1 locus and enabling the study of Topo I-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Topo I pathway restoration in tumor cells with silenced or reduced TOP1 expression.

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