RPE65 CRISPR/Cas9 KO Plasmid (h): sc-401489
- Target species: human
- 20 µg of transfection-ready, purified plasmid DNA; Suitable for up to 20 transfections
- RPE65 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 RPE65 genomic locus, resulting in gene knockout through non-homologous end joining (NHEJ)
- RPE65 HDR Plasmid (h) (sc-401489-HDR) is recommended for co-transfection with RPE65 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
- RPE65 HDR Plasmid (h) is a pool of plasmids, each containing a homology-directed repair (HDR) template corresponding to the gRNA target sites in the RPE65 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: RPE65 Antibody (E-5): sc-390787
Ordering Information
| Product Name | Catalog # | UNIT | Price | Qty | FAVORITES | |
RPE65 CRISPR/Cas9 KO Plasmid (h) | sc-401489 | 20 µg | $397.00 | |||
RPE65 HDR Plasmid (h) | sc-401489-HDR | 20 µg | $445.00 |
Overview
RPE65 encodes a retinoid isomerohydrolase expressed predominantly in retinal pigment epithelium, where it catalyzes the conversion of all-trans retinyl esters to 11-cis retinol in the visual (retinoid) cycle. This enzymatic step sustains regeneration of 11-cis retinal chromophore required for phototransduction and links retinoid metabolism to oxidative stress handling and membrane-associated lipid processing within the RPE. Perturbation of RPE65 function disrupts chromophore recycling, alters photoreceptor support, and drives downstream changes in gene programs associated with retinal homeostasis. Variants in RPE65 are implicated in inherited retinal dystrophies, making it a widely used target for studying mechanisms of retinal degeneration and retinoid pathway regulation.
RPE65 CRISPR/Cas9 KO Plasmid (h) is a pool of plasmids designed for targeted disruption of the RPE65 gene in human cell lines. Each plasmid in the pool co-expresses a unique sgRNA, targeting a distinct site within the RPE65 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, RPE65 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 RPE65 target site.
When co-transfected with RPE65 CRISPR/Cas9 KO Plasmid (h):
- The PuroR-RFP cassette integrates at the Cas9 cut site via HDR, disrupting the RPE65 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 RPE65 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 RPE65 exon(s) critical for RPE65 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.