IDO CRISPR/Cas9 KO Plasmid (h): sc-400495

Overview

Human IDO1 encodes indoleamine 2,3-dioxygenase (IDO), a heme-dependent enzyme that catalyzes the first and rate-limiting step of tryptophan degradation along the kynurenine pathway. By modulating local tryptophan availability and producing immunoregulatory metabolites, IDO1 influences inflammatory signaling, antigen-presenting cell function, and T cell responses downstream of cytokine and interferon-driven programs. IDO1 activity is frequently studied in the context of immune regulation within the tissue microenvironment, where metabolic reprogramming intersects with redox balance and stress-response pathways. Dysregulated IDO1 expression has been associated with chronic inflammation and tumor-associated immune suppression, making it a relevant node for mechanistic studies of immunometabolism.

IDO CRISPR/Cas9 KO Plasmid (h) is a pool of plasmids designed for targeted disruption of the IDO1 gene in human cell lines. Each plasmid in the pool co-expresses a unique sgRNA, targeting a distinct site within the IDO1 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, IDO 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 IDO1 target site.
When co-transfected with IDO CRISPR/Cas9 KO Plasmid (h):

• The PuroR-RFP cassette integrates at the Cas9 cut site via HDR, disrupting the IDO1 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 IDO1 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 IDO1 exon(s) critical for IDO 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.