RNase HII-A CRISPR Activation Plasmid (h): sc-405715-ACT

RNASEH2A encodes the catalytic subunit of ribonuclease H2, RNase HII-A, an endonuclease that recognizes and removes ribonucleotides misincorporated into genomic DNA and processes RNA:DNA hybrids. This activity supports ribonucleotide excision repair, preserves replication fork integrity, and limits replication-associated DNA damage signaling. RNASEH2A function is closely tied to genome maintenance pathways including DNA replication, S-phase checkpoint control, and resolution of R-loop–associated stress. Dysregulation of RNase H2 activity has been linked to inflammatory and genome instability phenotypes, making RNASEH2A a useful node for studying nucleic acid metabolism and DNA damage responses.

RNase HII-A CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous RNASEH2A expression without altering the underlying DNA sequence.

RNase HII-A CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the RNASEH2A 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 RNASEH2A transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous RNase HII-A expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native RNASEH2A locus and enabling the study of RNase HII-A-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of RNase HII-A pathway restoration in tumor cells with silenced or reduced RNASEH2A expression.

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