TS CRISPR/Cas9 KO Plasmid (h2): sc-402479-KO-2

Overview

Thymidylate synthase (TYMS; TS) is a cytosolic enzyme that catalyzes the reductive methylation of dUMP to dTMP using 5,10-methylenetetrahydrofolate, supplying thymidylate for DNA replication and repair. By linking folate-dependent one-carbon metabolism to de novo pyrimidine biosynthesis, TS helps maintain balanced dNTP pools and supports S-phase progression and genome stability. Altered TYMS expression or activity has been associated with replication stress responses and cell-cycle control in proliferative tissues. Dysregulation of this pathway is frequently studied in the context of tumor cell metabolism, DNA damage signaling, and nucleotide biosynthetic rewiring.

TS CRISPR/Cas9 KO Plasmid (h2) is a pool of plasmids designed for targeted disruption of the TYMS gene in human cell lines. Each plasmid in the pool co-expresses a unique sgRNA, targeting a distinct site within the TYMS 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, TS HDR Plasmid (h2) 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 TYMS target site.
When co-transfected with TS CRISPR/Cas9 KO Plasmid (h2):

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