RNase H1 CRISPR Activation Plasmid (h): sc-402850-ACT

Human RNASEH1 encodes RNase H1, a nuclear and mitochondrial endonuclease that specifically degrades the RNA strand of RNA:DNA hybrids, supporting removal of R-loops during transcription and replication. By processing RNA primers and resolving hybrid intermediates, RNase H1 contributes to genome stability, mitochondrial DNA replication and repair, and coordination of replication–transcription conflicts. Dysregulated RNA:DNA hybrid turnover has been linked to replication stress and DNA damage signaling, making RNASEH1 a relevant node for studying pathways that couple nucleic acid metabolism to cellular stress responses. Altered RNase H1 function is also associated with mitochondrial dysfunction phenotypes, enabling mechanistic investigation of mitochondrial genome maintenance in disease-relevant contexts.

RNase H1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous RNASEH1 expression without altering the underlying DNA sequence.

RNase H1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the RNASEH1 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 RNASEH1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous RNase H1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native RNASEH1 locus and enabling the study of RNase H1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of RNase H1 pathway restoration in tumor cells with silenced or reduced RNASEH1 expression.

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