PDIA2 CRISPR/Cas9 KO Plasmid (h): sc-413019

Overview

PDIA2 encodes protein disulfide isomerase family A member 2, an endoplasmic reticulum–associated oxidoreductase that supports oxidative protein folding by catalyzing disulfide bond formation and isomerization. Through its thioredoxin-like domains, PDIA2 contributes to proteostasis and quality control pathways linked to ER folding capacity, including processes that interface with the unfolded protein response and ER-associated degradation. Altered PDI family activity can modulate redox homeostasis and secretion-related phenotypes, making PDIA2 relevant for studying how folding enzymes shape cellular stress adaptation. Dysregulation of ER folding networks is implicated across conditions involving proteotoxic stress, such as cancer cell survival programs, neurodegeneration-associated protein misfolding, and inflammatory states driven by secretory pathway burden.

PDIA2 CRISPR/Cas9 KO Plasmid (h) is a pool of plasmids designed for targeted disruption of the PDIA2 gene in human cell lines. Each plasmid in the pool co-expresses a unique sgRNA, targeting a distinct site within the PDIA2 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, PDIA2 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 PDIA2 target site.
When co-transfected with PDIA2 CRISPR/Cas9 KO Plasmid (h):

• The PuroR-RFP cassette integrates at the Cas9 cut site via HDR, disrupting the PDIA2 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 PDIA2 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 PDIA2 exon(s) critical for PDIA2 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.