EphB2 CRISPR/Cas9 KO Plasmid (h): sc-401642

Overview

EPHB2 encodes EphB2, a receptor tyrosine kinase that binds ephrin-B ligands to mediate contact-dependent signaling important for cell positioning, tissue boundary formation, and axon guidance. EphB2 regulates cytoskeletal remodeling and adhesion through pathways involving Rho family GTPases, PI3K/AKT, MAPK/ERK, and crosstalk with WNT/β-catenin signaling, influencing cell migration and epithelial organization. Altered EPHB2 activity has been associated with dysregulated cell–cell communication and changes in invasive behavior in multiple cancer contexts, and it is also implicated in neurodevelopmental processes and synaptic function. These features make EphB2 a useful node for studying receptor-mediated signaling, polarity, and microenvironment-dependent regulation.

EphB2 CRISPR/Cas9 KO Plasmid (h) is a pool of plasmids designed for targeted disruption of the EPHB2 gene in human cell lines. Each plasmid in the pool co-expresses a unique sgRNA, targeting a distinct site within the EPHB2 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, EphB2 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 EPHB2 target site.
When co-transfected with EphB2 CRISPR/Cas9 KO Plasmid (h):

• The PuroR-RFP cassette integrates at the Cas9 cut site via HDR, disrupting the EPHB2 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 EPHB2 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 EPHB2 exon(s) critical for EphB2 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.