Not CRISPR data: the image illustrates antibody quality for visualizing CRISPR experiment results.
Not CRISPR data: the image illustrates antibody quality for visualizing CRISPR experiment results.
EEA1 CRISPR/Cas9 KO Plasmid (h): sc-400234
- Target species: human
- 20 µg of transfection-ready, purified plasmid DNA; Suitable for up to 20 transfections
- EEA1 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 EEA1 genomic locus, resulting in gene knockout through non-homologous end joining (NHEJ)
- EEA1 HDR Plasmid (h) (sc-400234-HDR) is recommended for co-transfection with EEA1 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
- EEA1 HDR Plasmid (h) is a pool of plasmids, each containing a homology-directed repair (HDR) template corresponding to the gRNA target sites in the EEA1 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: EEA1 Antibody (G-4): sc-137130
Ordering Information
| Product Name | Catalog # | UNIT | Price | Qty | FAVORITES | |
EEA1 CRISPR/Cas9 KO Plasmid (h) | sc-400234 | 20 µg | $397.00 | |||
EEA1 HDR Plasmid (h) | sc-400234-HDR | 20 µg | $445.00 |
Overview
EEA1 (early endosome antigen 1) is a Rab5 effector and PI3P-binding tether that coordinates early endosome docking and homotypic fusion through its FYVE domain and coiled-coil architecture. By organizing endosomal membrane identity and recruiting trafficking machinery, EEA1 regulates receptor-mediated endocytosis, endosome maturation, and downstream sorting decisions that shape signal transduction and nutrient uptake. Perturbation of EEA1-dependent trafficking can alter growth factor receptor recycling/degradation, antigen processing, and autophagy-linked membrane dynamics. Dysregulated endosomal pathways involving EEA1 have been implicated in cancer cell signaling plasticity, neurodegeneration-associated proteostasis stress, and pathogen entry/replication strategies that exploit the endocytic network.
EEA1 CRISPR/Cas9 KO Plasmid (h) is a pool of plasmids designed for targeted disruption of the EEA1 gene in human cell lines. Each plasmid in the pool co-expresses a unique sgRNA, targeting a distinct site within the EEA1 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, EEA1 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 EEA1 target site.
When co-transfected with EEA1 CRISPR/Cas9 KO Plasmid (h):
- The PuroR-RFP cassette integrates at the Cas9 cut site via HDR, disrupting the EEA1 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 EEA1 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 EEA1 exon(s) critical for EEA1 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.