CD223 CRISPR/Cas9 KO Plasmid (h): sc-406616

Overview

LAG3 (CD223) is an inhibitory immune checkpoint receptor expressed on activated T cells, regulatory T cells, NK cells, and subsets of dendritic cells, where it modulates antigen-driven activation and cytokine production. By engaging ligands such as MHC class II, LAG3 influences TCR signaling thresholds, T-cell exhaustion programs, and immune synapse dynamics, integrating with broader checkpoint and costimulatory networks. These pathways are frequently implicated in chronic infection and tumor-associated immune dysfunction, and LAG3 expression is widely used as a marker of dysfunctional or terminally differentiated T-cell states. Dysregulated LAG3 signaling and expression have also been linked to inflammatory and autoimmune-associated immune polarization, making it a relevant node for studying immune homeostasis.

CD223 CRISPR/Cas9 KO Plasmid (h) is a pool of plasmids designed for targeted disruption of the LAG3 gene in human cell lines. Each plasmid in the pool co-expresses a unique sgRNA, targeting a distinct site within the LAG3 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, CD223 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 LAG3 target site.
When co-transfected with CD223 CRISPR/Cas9 KO Plasmid (h):

• The PuroR-RFP cassette integrates at the Cas9 cut site via HDR, disrupting the LAG3 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 LAG3 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 LAG3 exon(s) critical for CD223 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.