RNase III Drosha CRISPR/Cas9 KO Plasmid (h): sc-401639
- Target species: human
- 20 µg of transfection-ready, purified plasmid DNA; Suitable for up to 20 transfections
- RNase III Drosha CRISPR/Cas9 Knockout (KO) Plasmid (h) is a pool of plasmids, each encoding Cas9 nuclease and a target-specific 20 nt guide RNA (gRNA) designed for maximum knockout efficiency using sequences derived from the GeCKO v2 library
- gRNA sequences direct Cas9 to induce site-specific double-strand breaks (DSBs) in the RNase III Drosha genomic locus, resulting in gene knockout through non-homologous end joining (NHEJ)
- RNase III Drosha HDR Plasmid (h) (sc-401639-HDR) is recommended for co-transfection with RNase III Drosha CRISPR/Cas9 KO Plasmid (h) to enable selection of successfully edited cells through HDR-mediated integration of a puromycin resistance cassette and RFP reporter gene
- RNase III Drosha HDR Plasmid (h) is a pool of plasmids, each containing a homology-directed repair (HDR) template corresponding to the gRNA target sites in the RNase III Drosha CRISPR/Cas9 KO Plasmid (h)
- Each HDR plasmid contains two ~800 bp homology arms flanking the puromycin resistance and RFP cassettes, designed to bind genomic DNA sequences surrounding the Cas9-induced double-strand break site and facilitate precise HDR-mediated integration
- The puromycin resistance and RFP genes are flanked by LoxP sites, enabling removal of selection markers via Cre recombinase (Cre Vector: sc-418923) after establishing stable knockout cell lines
- Following transfection, gene knockout efficiency can be assayed by WB, IF or IHC using antibody: RNase III Drosha Antibody (C-7): sc-393591
Ordering Information
| Product Name | Catalog # | UNIT | Price | Qty | FAVORITES | |
RNase III Drosha CRISPR/Cas9 KO Plasmid (h) | sc-401639 | 20 µg | $397.00 | |||
RNase III Drosha HDR Plasmid (h) | sc-401639-HDR | 20 µg | $445.00 |
Overview
DROSHA encodes RNase III Drosha, the nuclear endoribonuclease that initiates canonical microRNA biogenesis by cleaving primary miRNA transcripts into precursor miRNAs in concert with DGCR8, forming the Microprocessor complex. Through control of miRNA maturation, Drosha influences post-transcriptional gene regulation affecting cell-cycle progression, differentiation programs, and stress responses, with downstream impacts on pathways such as p53 signaling, TGF-β/SMAD, and innate immune regulation. Altered DROSHA activity or expression has been linked to dysregulated miRNA landscapes observed in multiple cancers and developmental disorders, and it is frequently studied in contexts of RNA processing defects and genome-wide expression remodeling. As a central node in RNA silencing and transcriptome homeostasis, Drosha is a key target for mechanistic studies of noncoding RNA regulation and disease-associated gene expression networks.
RNase III Drosha CRISPR/Cas9 KO Plasmid (h) is a pool of plasmids designed for targeted disruption of the DROSHA gene in human cell lines. Each plasmid in the pool co-expresses a unique sgRNA, targeting a distinct site within the DROSHA locus, alongside the Streptococcus pyogenes Cas9 nuclease, and encodes GFP to enable fluorescent identification and enrichment of successfully transfected cells. This multi-guide strategy increases the likelihood of inducing frameshifts or deletions that produce a functional knockout, offering a more robust alternative to single-guide approaches. DSBs induced at multiple sites are resolved through non-homologous end joining (NHEJ) or, when used with the included HDR donor template, homology-directed repair (HDR) at a defined target site within the locus.
When used in conjunction with the RFP-expressing HDR donor, GFP and RFP fluorescence can be used together to distinguish transfected from edited cell populations, streamlining flow cytometry-based sorting and clone selection workflows.
Homology-Directed Repair (HDR) Donor — Puromycin Cassette with RFP Reporter
For applications requiring confirmed, selectable knockout clones, RNase III Drosha HDR Plasmid (h) includes an HDR donor construct containing a puromycin resistance cassette (PuroR) and a red fluorescent protein (RFP) reporter, flanked by homology arms specific to a defined DROSHA target site.
When co-transfected with RNase III Drosha CRISPR/Cas9 KO Plasmid (h):
- The PuroR-RFP cassette integrates at the Cas9 cut site via HDR, disrupting the DROSHA open reading frame.
- RFP fluorescence provides an immediate visual indicator of successful integration, enabling fluorescence-based identification or sorting of edited cells prior to or alongside puromycin selection.
- Successfully edited cells are confirmed through puromycin resistance, substantially reducing clone screening burden.
- This selection strategy is ideal for generating stable, clonal KO cell lines for downstream functional studies, drug screening, or model development.
Cre-lox Cassette Removal System
The HDR donor construct features loxP sites flanking the PuroR-RFP selection cassette to allow clean marker removal following clone confirmation. Transient expression of Cre recombinase via the included Cre Vector: sc-418923 excises the cassette, leaving a minimal residual loxP site within the DROSHA locus and eliminating potential confounding effects on downstream assays.
This two-step approach:
- Minimizes disruption to local chromatin architecture and neighboring regulatory elements
- Restores a near-native genomic context at the edited locus
- Enables reuse of the puromycin selection strategy in the same cell line for additional edits
Key Features
- gRNA targeting DROSHA exon(s) critical for RNase III Drosha function
- Co-expression of SpCas9 and sgRNA from a single plasmid for simplified delivery
- HDR donor with puromycin resistance for positive clone selection
- loxP-flanked PuroR cassette with Cre recombinase vector for seamless marker removal
- Supplied ready to use for delivery by transfection
For Research Use Only. Not Intended for Diagnostic or Therapeutic Use.