RNase III Drosha CRISPR Activation Plasmid (h): sc-401639-ACT

DROSHA encodes the RNase III endonuclease Drosha, a core component of the nuclear Microprocessor complex that initiates canonical microRNA biogenesis by cleaving primary miRNA transcripts into precursor miRNAs. Through its cooperation with DGCR8 and downstream DICER processing, Drosha regulates post-transcriptional gene silencing programs that shape cell-cycle control, differentiation, stress responses, and innate signaling. Perturbation of DROSHA-dependent miRNA processing has been linked to widespread transcriptome dysregulation and altered DNA damage and apoptotic pathways. Aberrant Drosha activity or expression is associated with molecular phenotypes observed across multiple cancer and developmental disorder contexts, supporting its use as a mechanistic node for studying noncoding RNA networks.

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

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

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